Cell Mol Neurobiol (2015) 35:111–114 DOI 10.1007/s10571-014-0117-y
SHORT COMMUNICATION
Effect of Methylprednisolone on Mammalian Neuronal Networks In Vitro Matthias Wittstock • Paulus S. Rommer • Florian Schiffmann Konstantin Ju¨gelt • Simone Stu¨we • Reiner Benecke • Dietmar Schiffmann • Uwe K. Zettl
•
Received: 14 July 2014 / Accepted: 21 September 2014 / Published online: 5 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Glucocorticosteroids (GCS) are widely used for the treatment of neurological diseases, e.g. multiple sclerosis. High levels of GCS are toxic to the central nervous system and can produce adverse effects. The effect of methylprednisolone (MP) on mammalian neuronal networks was studied in vitro. We demonstrate a dosedependent excitatory effect of MP on cultured neuronal networks, followed by a shut-down of electrical activity using the microelectrode array technique. Keywords Neurological diseases Glucocorticosteroids Mammalian neuronal networks Neurotoxicity Electrical activity
Introduction Glucocorticosteroids (GCS) are widely used in the treatment of neurological diseases, e.g. multiple sclerosis (MS). Treatment effects of GCS in MS patients are due to antiDietmar Schiffmann—deceased. M. Wittstock (&) P. S. Rommer R. Benecke U. K. Zettl Department of Neurology, University of Rostock, Gehlsheimer Str. 20, 18147 Rostock, Germany e-mail: [email protected] P. S. Rommer e-mail: [email protected] P. S. Rommer Department of Neurology, Medical University of Vienna, Vienna, Austria F. Schiffmann K. Ju¨gelt S. Stu¨we D. Schiffmann Institute of Cell Biology and Biosystems Technology, University of Rostock, Rostock, Germany
inflammatory properties that are based on genomic and nongenomic mechanisms (Gold et al. 2001; Multiple Sclerosis Therapy Consensus Group (MSTCG) et al. 2008). Intrathecally administered GCS have shown antispastic effects in MS patients. However, those effects are not explained by the anti-inflammatory GCS properties as mostly progressive MS patients with no acute disease activity were treated (Abu-Mugheisib et al. 2011; Hellwig et al. 2004; Hoffmann et al. 2003, 2006). Additionally to clinical observations, neuronal hyperexcitability has been demonstrated functionally by patch-clamp technique (Foroutan et al. 1996). Little is known about the effect of GCS on neuronal networks. The aim of our study was to determine the dose-dependent effect of methylprednisolone (MP) in vitro on neuronal network activity and to analyse the functional impairment on the cellular network level by applying microelectrode array (MEA) technology.
Materials and Methods Cell Cultures Spinal cord (SC) tissue dissociated from 14 to 15 day old mouse embryos (crl:NMRI mice) was cultured according to the method of Ransom et al. (1977) with minor modifications including the use of DNAse and papain for tissue dissociation (Huettner and Baughan 1986). Cells were seeded on MEAs at concentrations of 250 K cells (neurons, glia cells, fibroblasts)/0.5 mL over a 600 mm2 surface of which 200 mm2 were activated for cell adhesion (10 mm2 over the recording matrix and 190 mm2 in a separate, isolated area designed to help for conditioning of the medium) (Gross and Kowlaski 1991). Most neuronal death occurred before 25 divisions, after which the cultures were
123
112
Cell Mol Neurobiol (2015) 35:111–114
between the amplifiers circuit board and the array at either side of the chamber to provide electrical contact with the recording matrix. During recording, networks were observed microscopically to monitor major neuronal stress or cell death. The multichannel neuronal data acquisition and recording system comprises two 32-channel pre-amplifiers (SMU, Texas, USA) positioned adjacent to the MEA and a 64-channel main amplifier (Alfa Electronics, Denton, Texas) including filtering and a 64-channel digital signal processor (University of Oldenburg, Oldenburg, Germany) that samples input signals at 12 bit and 20 kHz per channel. Control software (NEVpci, University of Oldenburg and Neuroexplorer, Plexon Inc., Denton, Texas) runs on a PC under Linux. Fig. 1 Experimental setting–neuronal network of SC cells cultured on MEA for 21 days
considered mature and stabilised (Fig. 1). Neurons were maintained for 1 week in MEM, containing 10 % foetal bovine serum and 10 % horse serum. Thereafter, cells were fed 2-times per week with MEM containing 10 % horse serum. When the glial carpet became confluent (approx. 5 days after seeding), glial growth was inhibited by the addition of 90–100 lM 5-Fluoro-2’-deoxyuridine ? uridine for 48 h. The cultures were maintained at 37 °C in an atmosphere containing 10 % CO2. Microelectrode Arrays The MEAs were fabricated by applying standard photoetching technique. This technique and the concomitant culture methods have been described previously (Gramowski et al. 2000; Gross and Kowlaski 1991; Huettner and Baughan 1986). Briefly, commercially available, sputtered indium tin oxide (ITO) plates were photo etched, cut into 5 9 5 cm waves, spin-insulated with polysiloxane, cured, de-insulated at the electrode tips with single laser shots, and electrolytically gold-plated to adjust the interface of the exposed ITO to approximately 1 mOhm. The hydrophobic insulation material required surface activation by a brief pulse from a propane flame. Flaming through masks allowed the creation of coarse patterns of adhesive regions such as 1–4 diameter islands centred on the electrode array. Poly-D-lysine (25 lg/mL; 30–70 kD) and laminin (16 lg/ mL) were added to the activated surface regions. The recording chamber (Gross 1994) consisted of an aluminium plate holding the MEA, a stainless steel chamber cover with microscope port, and a small plastic cap that confined a 10 % CO2 atmosphere maintained by humidified flow at approximately 15 mL/min. Two zebra strips (carbon-filled silicone elastomere, Fujipoly, Cranford, NJ) were pressed
123
Methylprednisolone Preparation and Evaluation of SC Network Response to MP MP was purchased as UrbasonÓ (Sanofi Pharma, Bad Soden am Taunus, Germany). It was diluted with distilled sterile water to get a stock solution of 50 mg MP/1 mL, which was further diluted to get indicated final concentrations. Because UrbasonÓ-solution is unstable after 2 days (Adventis Pharma), it was used for experiments within 24 h. After an initial 30 min recording period to determine the nature of the native activity, MP was added to the culture medium to yield final concentrations of 10, 30, 50, 75, 100, 150, 200 and 250 lg/mL, respectively, and incubated at 37 °C. The MP concentrations correspond to a molarity of 0.0267–0.667 mM. The electrical activity was recorded and documented as spikes/min and bursts/min., resp., for 30 min (spikes from active channels were integrated, and displayed on chart records and visually inspected for baseline, spike and burst activity). Apoptosis was assessed according to the morphological changes as well as to in situ nick translation and tailing (Gold et al. 1994; Zettl et al. 1995). Statistical Analysis The data of the experiments were subjected to analysis of significance by a Students t test. Significance was concluded for p \ 0.05. Least squares means and standard errors (SE) are presented in the graphs.
Results The data of six experiments were analysed. The SC cells were cultivated for 39 ± 8 days on MEAs. The experiments showed a clear dose-dependent excitatory effect of MP on MEA recorded SC cell function. The effect
Cell Mol Neurobiol (2015) 35:111–114
113
occurred within 5 min after adding the MP solution. Spiking rate and burst firing were augmented, when increasing MP concentrations were added between 10 and 150 lg/mL to the cultures. Higher concentrations caused an irreversible shut-off. The minimum shut-off concentration was determined as 208 lg/mL MP ± 74. The augmentation effect was reversible by complete medium change in a range from 100 to 200 lg/mL MP. At higher MP concentrations the effect was not reversible. Osmolarity was maintained between 334 and 412 mM (Fig. 2).
*
spikes/min
A 3000 2500 2000 1500 1000 500 0
native
10
30
50
75
100
150
200
250
methyl-prednisolone [µg/mL]
B
*
140
*
*
bursts/min
120 100 80 60 40 20 0
native
10
30
50
75
100
150
200
250
methyl-prednisolone [µg/mL]
C
Fig. 2 Dose-dependent spike and bursting activity in response to MP. a Increasing spike activity with increasing MP concentrations and shut-off at MP concentrations higher than 208 lg/mL (mean value of 6 experiments) as recorded on MEA 30 min after MP application (spikes per minute). Native represents recording without MP. Least squares means and standard errors (SE) are presented in the graphs (*p \ 0.05). b Increasing burst activity with increasing MP concentrations and shut-off at MP concentrations higher than 208 lg/mL (mean value of 6 experiments) as recorded on MEA 30 min after MP application (bursts per minute). Native represents recording without MP. Least squares means and standard errors (SE) are presented in the graphs (*p \ 0.05). c Example of digital burst integration: bursts are defined using two thresholds (T1) to determine burst starting and stopping time. The latter represents a relative threshold that selects the bursts for analysis; ba burst amplitude, bd burst duration
Discussion Reports on MP treatment in the escalating immunotherapy of MS (Multiple Sclerosis Therapy Consensus Group (MSTCG) et al. 2008; Zivadinov et al. 2001) focused on dose-dependent immune-modulating effects (Gold et al. 2001). Recently, symptom-associated effects of GCS (e.g. reduction of spasticity) were reported (Abu-Mugheisib et al. 2011; Hellwig et al. 2004; Hoffmann et al. 2003, 2006). Our study aimed to explore the effects of electrophysiological properties of GCS. Previously, the effects of an intensive GCS (e.g. MP) regimen have been studied on the electrical properties of cat lumbar spinal motor neurons via intracellular recording. The effects of GCS on GABA and glutamate channels have been characterised by Foroutan et al. (1996); excitatory effects have been well described and will outweigh inhibitory ones. Sasaki et al. could show dose-dependent effects of MP on SC axons in vitro. The effects were due to modulation of 8-OH-DPAT-induced amplitude depression, that may act as an agonist for the 5-HT1A and 5-HT7 receptors as well as an serotonin reuptake inhibiting/ releasing agent (Sasaki et al. 2002). The serotonergic system has been well described in neuropsychiatry. Further effects on the CNS and on the autonomous nervous systems remain at least partly unclear. The effects of GCS on neuron ionic channels are complex, and the aim of the study was to demonstrate whether in vitro effects may be in line with treatment observations in humans. The reported results showed that GCS at different doses produced different effects on motor neuron excitability, impulse generation and conduction. An additional effect of GCS was augmentation of the action potential after-depolarization. GCS have complex effects on specific ionic mechanisms in the CNS (Hall 1982) that are hitherto not fully understood. To our knowledge, we demonstrated for the first time a dose-dependent excitatory effect of MP in cultured neuronal networks, followed by a shut-down of electric activity at higher MP concentration than 200 lg/mL. The effect of MP appears as early as 5 min after adding MP to the culture. The effects of high-dose MP therapy are suggested to be mediated by a nongenomic membrane activity with rapid onset (Gold et al. 2001). In vitro observations demonstrated that MP increases the frequency and the amplitude of excitatory postsynaptic currents as well as the frequency of inhibitory postsynaptic currents. Nevertheless, the amplitude of inhibitory postsynaptic currents is decreased (Foroutan et al. 1996). With high MP concentration, the excitatory effects will diminish in vitro. Cell apoptosis and necrosis were not significant in our experiment. Consequently, the shut-off is not due to cell death.
123
114
The concentrations of GCS intrathecally administered in the CSF of MS patients range from 200 to 666 lg/ml. Thus, in vitro observations may suggest a GCS dosedependent anti-spastic effect in humans. The used experimental setting may be a promising tool in the evaluation of further potential neuroactive therapeutical agents avoiding the use of animal models. To elucidate the effects of GCS in humans, the next steps might be the assessment of activity of the different channels in humans.
Conflict of interest All authors state no potential conflict with the trial, and the authors have not received any financial or material support in conducting this trial.
References Abu-Mugheisib M, Benecke R, Zettl UK (2011) Repeated intrathecal triamcinolone acetonide administration in progressive multiple sclerosis: a review. Mult Scler Int 2011:219049 Foroutan A, Behbehan MM, Anderson DL (1996) Effects of methylprednisolone on the GABA-and glutamate-induced currents: relevance to glucocorticoid-induced neurotoxicity and brain aging. Steroids 61:354–366 Gold R, Schmied M, Giegerich G, Breitschopf H, Hartung HP, Toyka KV, Lassmann H (1994) Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest 71(2):219–225 Gold R, Buttgereit F, Toyka KV (2001) Mechanism of action of glucocorticosteroid hormones: possible implications for therapy of neuroimmunlogical disorders. J Neuroimmunol 117:1–8 Gramowski A, Schiffmann D, Gross GW (2000) Quantification of acute neurotoxic effects of trimethyltin using neuronal networks cultured on microelectrode arrays. Neuro Toxicol 2:331–342 Gross GW (1994) Internal dynamics of randomized mammalian neuronal networks in culture. In: Stenger DA, McKenna TM (eds) Enabling technologies for cultured neuronal networks. Academic press, New York, pp 277–317
123
Cell Mol Neurobiol (2015) 35:111–114 Gross GW, Kowlaski JM (1991) Experimental and theoretical analysis of random nerve cell network dynamics. In: Antogentti P, Milutinovic V (eds) Neuronal networks: concepts, applications, and implementations. Prentice-Hall Inc, Englewood, pp 47–110 Hall ED (1982) Glucocorticoid effects on the electrical properties of spinal motor neurons. Brain Res 240:109–116 Hellwig K, Stein FJ, Przuntek H, Mu¨ller T (2004) Efficacy of repeated intrathecal triamcinolone acetonide application in progressive multiple sclerosis patients with spinal symptoms. BMC Neurol 4(1):18 Hoffmann V, Schimriegk S, Isalmova S, Hellwig K, Lukas C, Brune N, Po¨hlau D, Przuntek H, Mu¨ller T (2003) Efficacy and safety of repeated intrathecal triamcinolone acetonide application in progressive multiple sclerosis patients. J Neurol Sci 211:81–84 Hoffmann V, Kuhn W, Schimrigk S, Islamova S, Hellwig K, Lukas C, Brune N, Po¨hlau D, Przuntek H, Mu¨ller T (2006) Repeat intrathecal triamcinolone acetonide application is beneficial in progressive MS patients. Eur J Neurol 13(1):72–76 Huettner JE, Baughan RW (1986) Primary culture of identified neurons from the visual cortex of postnatal rats. J Neurosci 6:3044–3060 Multiple Sclerosis Therapy Consensus Group (MSTCG), Wiendl H, Toyka KV, Rieckmann P, Gold R, Hartung HP, Hohlfeld R (2008) Basic and escalating immunomodulatory treatments in multiple sclerosis: current therapeutic recommendations. J Neurol 255(10):1449–1463 Ransom BR, Neale E, Henkart M, Bullock PN, Nelson PG (1977) Mouse spinal cord in cell culture. I. Morphology and intrisic neuronal electrophysiologic properties. J Neurophysiol 40:1132–1150 Sasaki T, Sakuma J, Ichikawa T, Matsumoto M, Tiwari P, Young W, Kodama N (2002) Effects of methylprednisolone on axonal depression induced by hypoxia, gamma-aminobutyric acid, and (±)-8-hydroxy-dipropylaminotetralin hydrobromide. Neurosurgery 51(6):1477–1483 Zettl UK, Gold R, Toyka KV, Hartung HP (1995) Intravenous glucocorticosteroid treatment augments apoptosis of inflammatory T cells in experimental autoimmune neuritis (EAN) of the Lewis rat. J Neuropathol Exp Neurol 54(4):540–547 Zivadinov R, Rudick RA, De Masi R, Nasuelli D, Ukmar M, PozziMucelli RS, Grop A, Cazzato G, Zorzon M (2001) Effects of intravenous methylprednisolone on brain atrophy in relapsingremitting MS. Neurology 57:1239–1247
SHORT COMMUNICATION
Effect of Methylprednisolone on Mammalian Neuronal Networks In Vitro Matthias Wittstock • Paulus S. Rommer • Florian Schiffmann Konstantin Ju¨gelt • Simone Stu¨we • Reiner Benecke • Dietmar Schiffmann • Uwe K. Zettl
•
Received: 14 July 2014 / Accepted: 21 September 2014 / Published online: 5 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Glucocorticosteroids (GCS) are widely used for the treatment of neurological diseases, e.g. multiple sclerosis. High levels of GCS are toxic to the central nervous system and can produce adverse effects. The effect of methylprednisolone (MP) on mammalian neuronal networks was studied in vitro. We demonstrate a dosedependent excitatory effect of MP on cultured neuronal networks, followed by a shut-down of electrical activity using the microelectrode array technique. Keywords Neurological diseases Glucocorticosteroids Mammalian neuronal networks Neurotoxicity Electrical activity
Introduction Glucocorticosteroids (GCS) are widely used in the treatment of neurological diseases, e.g. multiple sclerosis (MS). Treatment effects of GCS in MS patients are due to antiDietmar Schiffmann—deceased. M. Wittstock (&) P. S. Rommer R. Benecke U. K. Zettl Department of Neurology, University of Rostock, Gehlsheimer Str. 20, 18147 Rostock, Germany e-mail: [email protected] P. S. Rommer e-mail: [email protected] P. S. Rommer Department of Neurology, Medical University of Vienna, Vienna, Austria F. Schiffmann K. Ju¨gelt S. Stu¨we D. Schiffmann Institute of Cell Biology and Biosystems Technology, University of Rostock, Rostock, Germany
inflammatory properties that are based on genomic and nongenomic mechanisms (Gold et al. 2001; Multiple Sclerosis Therapy Consensus Group (MSTCG) et al. 2008). Intrathecally administered GCS have shown antispastic effects in MS patients. However, those effects are not explained by the anti-inflammatory GCS properties as mostly progressive MS patients with no acute disease activity were treated (Abu-Mugheisib et al. 2011; Hellwig et al. 2004; Hoffmann et al. 2003, 2006). Additionally to clinical observations, neuronal hyperexcitability has been demonstrated functionally by patch-clamp technique (Foroutan et al. 1996). Little is known about the effect of GCS on neuronal networks. The aim of our study was to determine the dose-dependent effect of methylprednisolone (MP) in vitro on neuronal network activity and to analyse the functional impairment on the cellular network level by applying microelectrode array (MEA) technology.
Materials and Methods Cell Cultures Spinal cord (SC) tissue dissociated from 14 to 15 day old mouse embryos (crl:NMRI mice) was cultured according to the method of Ransom et al. (1977) with minor modifications including the use of DNAse and papain for tissue dissociation (Huettner and Baughan 1986). Cells were seeded on MEAs at concentrations of 250 K cells (neurons, glia cells, fibroblasts)/0.5 mL over a 600 mm2 surface of which 200 mm2 were activated for cell adhesion (10 mm2 over the recording matrix and 190 mm2 in a separate, isolated area designed to help for conditioning of the medium) (Gross and Kowlaski 1991). Most neuronal death occurred before 25 divisions, after which the cultures were
123
112
Cell Mol Neurobiol (2015) 35:111–114
between the amplifiers circuit board and the array at either side of the chamber to provide electrical contact with the recording matrix. During recording, networks were observed microscopically to monitor major neuronal stress or cell death. The multichannel neuronal data acquisition and recording system comprises two 32-channel pre-amplifiers (SMU, Texas, USA) positioned adjacent to the MEA and a 64-channel main amplifier (Alfa Electronics, Denton, Texas) including filtering and a 64-channel digital signal processor (University of Oldenburg, Oldenburg, Germany) that samples input signals at 12 bit and 20 kHz per channel. Control software (NEVpci, University of Oldenburg and Neuroexplorer, Plexon Inc., Denton, Texas) runs on a PC under Linux. Fig. 1 Experimental setting–neuronal network of SC cells cultured on MEA for 21 days
considered mature and stabilised (Fig. 1). Neurons were maintained for 1 week in MEM, containing 10 % foetal bovine serum and 10 % horse serum. Thereafter, cells were fed 2-times per week with MEM containing 10 % horse serum. When the glial carpet became confluent (approx. 5 days after seeding), glial growth was inhibited by the addition of 90–100 lM 5-Fluoro-2’-deoxyuridine ? uridine for 48 h. The cultures were maintained at 37 °C in an atmosphere containing 10 % CO2. Microelectrode Arrays The MEAs were fabricated by applying standard photoetching technique. This technique and the concomitant culture methods have been described previously (Gramowski et al. 2000; Gross and Kowlaski 1991; Huettner and Baughan 1986). Briefly, commercially available, sputtered indium tin oxide (ITO) plates were photo etched, cut into 5 9 5 cm waves, spin-insulated with polysiloxane, cured, de-insulated at the electrode tips with single laser shots, and electrolytically gold-plated to adjust the interface of the exposed ITO to approximately 1 mOhm. The hydrophobic insulation material required surface activation by a brief pulse from a propane flame. Flaming through masks allowed the creation of coarse patterns of adhesive regions such as 1–4 diameter islands centred on the electrode array. Poly-D-lysine (25 lg/mL; 30–70 kD) and laminin (16 lg/ mL) were added to the activated surface regions. The recording chamber (Gross 1994) consisted of an aluminium plate holding the MEA, a stainless steel chamber cover with microscope port, and a small plastic cap that confined a 10 % CO2 atmosphere maintained by humidified flow at approximately 15 mL/min. Two zebra strips (carbon-filled silicone elastomere, Fujipoly, Cranford, NJ) were pressed
123
Methylprednisolone Preparation and Evaluation of SC Network Response to MP MP was purchased as UrbasonÓ (Sanofi Pharma, Bad Soden am Taunus, Germany). It was diluted with distilled sterile water to get a stock solution of 50 mg MP/1 mL, which was further diluted to get indicated final concentrations. Because UrbasonÓ-solution is unstable after 2 days (Adventis Pharma), it was used for experiments within 24 h. After an initial 30 min recording period to determine the nature of the native activity, MP was added to the culture medium to yield final concentrations of 10, 30, 50, 75, 100, 150, 200 and 250 lg/mL, respectively, and incubated at 37 °C. The MP concentrations correspond to a molarity of 0.0267–0.667 mM. The electrical activity was recorded and documented as spikes/min and bursts/min., resp., for 30 min (spikes from active channels were integrated, and displayed on chart records and visually inspected for baseline, spike and burst activity). Apoptosis was assessed according to the morphological changes as well as to in situ nick translation and tailing (Gold et al. 1994; Zettl et al. 1995). Statistical Analysis The data of the experiments were subjected to analysis of significance by a Students t test. Significance was concluded for p \ 0.05. Least squares means and standard errors (SE) are presented in the graphs.
Results The data of six experiments were analysed. The SC cells were cultivated for 39 ± 8 days on MEAs. The experiments showed a clear dose-dependent excitatory effect of MP on MEA recorded SC cell function. The effect
Cell Mol Neurobiol (2015) 35:111–114
113
occurred within 5 min after adding the MP solution. Spiking rate and burst firing were augmented, when increasing MP concentrations were added between 10 and 150 lg/mL to the cultures. Higher concentrations caused an irreversible shut-off. The minimum shut-off concentration was determined as 208 lg/mL MP ± 74. The augmentation effect was reversible by complete medium change in a range from 100 to 200 lg/mL MP. At higher MP concentrations the effect was not reversible. Osmolarity was maintained between 334 and 412 mM (Fig. 2).
*
spikes/min
A 3000 2500 2000 1500 1000 500 0
native
10
30
50
75
100
150
200
250
methyl-prednisolone [µg/mL]
B
*
140
*
*
bursts/min
120 100 80 60 40 20 0
native
10
30
50
75
100
150
200
250
methyl-prednisolone [µg/mL]
C
Fig. 2 Dose-dependent spike and bursting activity in response to MP. a Increasing spike activity with increasing MP concentrations and shut-off at MP concentrations higher than 208 lg/mL (mean value of 6 experiments) as recorded on MEA 30 min after MP application (spikes per minute). Native represents recording without MP. Least squares means and standard errors (SE) are presented in the graphs (*p \ 0.05). b Increasing burst activity with increasing MP concentrations and shut-off at MP concentrations higher than 208 lg/mL (mean value of 6 experiments) as recorded on MEA 30 min after MP application (bursts per minute). Native represents recording without MP. Least squares means and standard errors (SE) are presented in the graphs (*p \ 0.05). c Example of digital burst integration: bursts are defined using two thresholds (T1) to determine burst starting and stopping time. The latter represents a relative threshold that selects the bursts for analysis; ba burst amplitude, bd burst duration
Discussion Reports on MP treatment in the escalating immunotherapy of MS (Multiple Sclerosis Therapy Consensus Group (MSTCG) et al. 2008; Zivadinov et al. 2001) focused on dose-dependent immune-modulating effects (Gold et al. 2001). Recently, symptom-associated effects of GCS (e.g. reduction of spasticity) were reported (Abu-Mugheisib et al. 2011; Hellwig et al. 2004; Hoffmann et al. 2003, 2006). Our study aimed to explore the effects of electrophysiological properties of GCS. Previously, the effects of an intensive GCS (e.g. MP) regimen have been studied on the electrical properties of cat lumbar spinal motor neurons via intracellular recording. The effects of GCS on GABA and glutamate channels have been characterised by Foroutan et al. (1996); excitatory effects have been well described and will outweigh inhibitory ones. Sasaki et al. could show dose-dependent effects of MP on SC axons in vitro. The effects were due to modulation of 8-OH-DPAT-induced amplitude depression, that may act as an agonist for the 5-HT1A and 5-HT7 receptors as well as an serotonin reuptake inhibiting/ releasing agent (Sasaki et al. 2002). The serotonergic system has been well described in neuropsychiatry. Further effects on the CNS and on the autonomous nervous systems remain at least partly unclear. The effects of GCS on neuron ionic channels are complex, and the aim of the study was to demonstrate whether in vitro effects may be in line with treatment observations in humans. The reported results showed that GCS at different doses produced different effects on motor neuron excitability, impulse generation and conduction. An additional effect of GCS was augmentation of the action potential after-depolarization. GCS have complex effects on specific ionic mechanisms in the CNS (Hall 1982) that are hitherto not fully understood. To our knowledge, we demonstrated for the first time a dose-dependent excitatory effect of MP in cultured neuronal networks, followed by a shut-down of electric activity at higher MP concentration than 200 lg/mL. The effect of MP appears as early as 5 min after adding MP to the culture. The effects of high-dose MP therapy are suggested to be mediated by a nongenomic membrane activity with rapid onset (Gold et al. 2001). In vitro observations demonstrated that MP increases the frequency and the amplitude of excitatory postsynaptic currents as well as the frequency of inhibitory postsynaptic currents. Nevertheless, the amplitude of inhibitory postsynaptic currents is decreased (Foroutan et al. 1996). With high MP concentration, the excitatory effects will diminish in vitro. Cell apoptosis and necrosis were not significant in our experiment. Consequently, the shut-off is not due to cell death.
123
114
The concentrations of GCS intrathecally administered in the CSF of MS patients range from 200 to 666 lg/ml. Thus, in vitro observations may suggest a GCS dosedependent anti-spastic effect in humans. The used experimental setting may be a promising tool in the evaluation of further potential neuroactive therapeutical agents avoiding the use of animal models. To elucidate the effects of GCS in humans, the next steps might be the assessment of activity of the different channels in humans.
Conflict of interest All authors state no potential conflict with the trial, and the authors have not received any financial or material support in conducting this trial.
References Abu-Mugheisib M, Benecke R, Zettl UK (2011) Repeated intrathecal triamcinolone acetonide administration in progressive multiple sclerosis: a review. Mult Scler Int 2011:219049 Foroutan A, Behbehan MM, Anderson DL (1996) Effects of methylprednisolone on the GABA-and glutamate-induced currents: relevance to glucocorticoid-induced neurotoxicity and brain aging. Steroids 61:354–366 Gold R, Schmied M, Giegerich G, Breitschopf H, Hartung HP, Toyka KV, Lassmann H (1994) Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest 71(2):219–225 Gold R, Buttgereit F, Toyka KV (2001) Mechanism of action of glucocorticosteroid hormones: possible implications for therapy of neuroimmunlogical disorders. J Neuroimmunol 117:1–8 Gramowski A, Schiffmann D, Gross GW (2000) Quantification of acute neurotoxic effects of trimethyltin using neuronal networks cultured on microelectrode arrays. Neuro Toxicol 2:331–342 Gross GW (1994) Internal dynamics of randomized mammalian neuronal networks in culture. In: Stenger DA, McKenna TM (eds) Enabling technologies for cultured neuronal networks. Academic press, New York, pp 277–317
123
Cell Mol Neurobiol (2015) 35:111–114 Gross GW, Kowlaski JM (1991) Experimental and theoretical analysis of random nerve cell network dynamics. In: Antogentti P, Milutinovic V (eds) Neuronal networks: concepts, applications, and implementations. Prentice-Hall Inc, Englewood, pp 47–110 Hall ED (1982) Glucocorticoid effects on the electrical properties of spinal motor neurons. Brain Res 240:109–116 Hellwig K, Stein FJ, Przuntek H, Mu¨ller T (2004) Efficacy of repeated intrathecal triamcinolone acetonide application in progressive multiple sclerosis patients with spinal symptoms. BMC Neurol 4(1):18 Hoffmann V, Schimriegk S, Isalmova S, Hellwig K, Lukas C, Brune N, Po¨hlau D, Przuntek H, Mu¨ller T (2003) Efficacy and safety of repeated intrathecal triamcinolone acetonide application in progressive multiple sclerosis patients. J Neurol Sci 211:81–84 Hoffmann V, Kuhn W, Schimrigk S, Islamova S, Hellwig K, Lukas C, Brune N, Po¨hlau D, Przuntek H, Mu¨ller T (2006) Repeat intrathecal triamcinolone acetonide application is beneficial in progressive MS patients. Eur J Neurol 13(1):72–76 Huettner JE, Baughan RW (1986) Primary culture of identified neurons from the visual cortex of postnatal rats. J Neurosci 6:3044–3060 Multiple Sclerosis Therapy Consensus Group (MSTCG), Wiendl H, Toyka KV, Rieckmann P, Gold R, Hartung HP, Hohlfeld R (2008) Basic and escalating immunomodulatory treatments in multiple sclerosis: current therapeutic recommendations. J Neurol 255(10):1449–1463 Ransom BR, Neale E, Henkart M, Bullock PN, Nelson PG (1977) Mouse spinal cord in cell culture. I. Morphology and intrisic neuronal electrophysiologic properties. J Neurophysiol 40:1132–1150 Sasaki T, Sakuma J, Ichikawa T, Matsumoto M, Tiwari P, Young W, Kodama N (2002) Effects of methylprednisolone on axonal depression induced by hypoxia, gamma-aminobutyric acid, and (±)-8-hydroxy-dipropylaminotetralin hydrobromide. Neurosurgery 51(6):1477–1483 Zettl UK, Gold R, Toyka KV, Hartung HP (1995) Intravenous glucocorticosteroid treatment augments apoptosis of inflammatory T cells in experimental autoimmune neuritis (EAN) of the Lewis rat. J Neuropathol Exp Neurol 54(4):540–547 Zivadinov R, Rudick RA, De Masi R, Nasuelli D, Ukmar M, PozziMucelli RS, Grop A, Cazzato G, Zorzon M (2001) Effects of intravenous methylprednisolone on brain atrophy in relapsingremitting MS. Neurology 57:1239–1247
