logy 550 (2006) 33–38www.elsevier.com/locate/ejphar

European Journal of Pharmaco

Baclofen and potential therapeutic use: Studies of neuronal survival

Alessandro Dario a,1, Rossana Pisani b,⁎,1, Simone Sangiorgi a, Andrea Soragna b,d,Marcella Reguzzoni c, Marina Protasoni c, Federico Pessina a, Riccardo Fesce b,

Antonio Peres b, Giustino Tomei a

a Neurosurgical Clinic, University of Insubria, Via L. Borri 57, 21100 Varese, Italyb Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology, and Center for Neurosciences,

University of Insubria, Via Dunant 3, 21100 Varese, Italyc Department of Human Morphology, University of Insubria, Via M.te Generoso 71, 21100 Varese, Italy

d Institute of General Physiology and Biological Chemistry, University of Milan, Via Trentacoste, 2, 20134 Milano, Italy

Received 4 April 2006; received in revised form 21 August 2006; accepted 23 August 2006Available online 8 September 2006

Abstract

Up to now, baclofen (a GABAB receptor agonist) has been used for the treatment of severe spasticity unresponsive to oral antispasmodics.Although in humans it is usually administered at 2 mg/ml, the dosage to be used in the treatment of other diseases is unknown. For this reason, it isimportant to determine the safe maximum dosage and toxicity at the clinically used concentration. Primary cortical neurons represent a usefulmodel to test the safety of baclofen. We performed a colorimetric assay (MTT test) as well as electron microscopy investigations, to determineneuronal survival after the treatment with baclofen at a concentration of 2 and 4 mg/ml. Our results demonstrated that, in our experimental model,neither concentration affected neuronal survival. Considering the above results, we can conclude that at the used concentrations, this drug is safeand its clinical use should be encouraged.© 2006 Elsevier B.V. All rights reserved.

Keywords: Colorimetric assay; Primary cortical neurons; Baclofen; Neuronal survival; Spasticity

1. Introduction

Baclofen (β-p-clorofenil-GABA, commercial name Liore-sal®, produced by Novartis Pharma) is a γ-aminobutyric acid(GABA) agonist acting on metabotropic GABAB receptors. Itseffect is due to a balance between inhibition of neurotransmitterrelease, mediated by presynaptic GABAB receptors, andinhibition of neuronal excitability, mediated by the postsynapticGABAA receptors (Misgeld et al., 1995).

Within the mammalian brain, the highest density of GABAB

binding sites is found in the thalamic nuclei, themolecular layer ofthe cerebellum, the cerebral cortex, the interpeduncular nucleus,and the dorsal horn of the spinal cord (Bowery et al., 1987).

⁎ Corresponding author. Department of Structural and Functional Biology,University of Insubria, Via Dunant 3, 21100 Varese, Italy. Tel.: +39 0332421318; fax: +39 0332 421300.

E-mail address: rossana.pisani@uninsubria.it (R. Pisani).1 These authors contributed equally.

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.08.068

From a molecular point of view, GABAB agonists inhibitbasal and forskolin-stimulated neuronal adenylate cyclase inbrain slices through a G protein-dependent mechanism, resultingin a reduced level of intracellular cAMP (Knight and Bowery,1996). Interestingly, this effect is blocked by pertussis toxin,which suggests the involvement of Gi/o rather than Gs protein(Harayama et al., 1998). Baclofen binding to GABAB receptors,which are reported to be coupled to G proteins, modulatesvoltage-gated ion channels (e.g. calcium and potassium) (Carterand Mynlieff, 2004; Moran et al., 2004). In particular, theactivation of the potassium conductance produces hyperpolar-isation of the cell membrane, leading to long-lasting inhibitorypostsynaptic potentials and a reduction in neuronal excitability(Bussieres and El Manira, 1999; Harayama et al., 1998).Furthermore, while the effect on the Ca2+ conductance appearsto be primarily associated with presynaptic P/Q- and N-typecurrents, the modulation of the K+ conductance appears to belinked primarily with postsynaptic GABAB sites (Bowery et al.,2002).

34 A. Dario et al. / European Journal of Pharmacology 550 (2006) 33–38

Besides the cited cellular effects, activation of this receptorleads to a plethora of both hormonal and behavioural effects.GABAB receptor agonists have a number of pharmacologicaleffects in the central nervous system: central muscle relaxation,suppression of cocaine and narcotic drug self-administration,antinociception, cognitive impairment, inhibition of hormonerelease, synaptic plasticity, development of some neuronal path-ways, as well as improvement of conduction in demyelinatedaxons (Bowery et al., 2002).

The centrally mediated muscle relaxant effect of baclofen isthe most widely exploited clinical property of the drug (Orsneset al., 2000). This action appears to be due to baclofen-inducedreduction in neurotransmitter release onto motoneurons in theventral horn of the spinal cord. There is also a suggestion thatthe antispastic effect is due to a post- rather than a presynapticaction on motoneurons (Orsnes et al., 2000). Regardless of thesite of action, the efficacy of baclofen in alleviating spasticityhas made it the drug of choice for this condition (Bowery et al.,2002). Baclofen is effective in treating spasticity associatedwith tardive dystonia, brain and spinal cord injury, cerebralpalsy, tetanus, multiple sclerosis, and stiff-man syndrome(Becker et al., 2000; Krach, 2001). Given the above mentionedtherapeutic effects, the future or possible clinical applications ofbaclofen could be numerous, as suggested by experimentalstudies of movement disorders, cerebral ischaemia, neuroplas-ticity, epileptogenesis, panic attack, catatonia, and pain (Boweryet al., 2002). Since baclofen is usually administered intrathe-cally, given its poor ability to cross the blood–brain barrierwhen administered orally, and considering that the best dosagefor the treatment of the above cited diseases is not yet known,the first step to validate the administration of intrathecalbaclofen for new indications, would be to test the safety of theclinically used concentration (2 mg/ml) and to identify the safemaximum dosage on cortical neurons (which have a highexpression of GABAB receptors (Bowery et al., 1987)). A studyaimed at evaluating the eventual toxicity of baclofen appearstherefore to be important, especially because local anaestheticsadministered intratechally have been shown to be neurotoxic(Radwan et al., 2002).

To do this, we performed a colorimetric assay (MTT test,(Mosmann, 1983)), as well as electron microscopy investiga-tions, to determine the survival of primary cortical neurons aftertreatment with baclofen.

2. Materials and methods

2.1. Drugs and chemicals

Media and sera were obtained from Invitrogen (San GiulianoMilanese, Italy). (RS)-Baclofen was from Tocris Biosciences(Avonmouth, UK). It was initially dissolved in HCl 1N to aconcentration of 250 mg/ml and further diluted with platingmedium to the desired concentration. Lioresal® was fromNovartis Pharma (Origgio, Italy). It was diluted to the desiredconcentration with plating medium. MTT ([3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide]) and all otherchemicals were purchased from Sigma Aldrich (St. Louis, Mo).

2.2. Cortical neurons culture

Primary neuronal cultures were prepared as described indetail elsewhere with minor modifications (Bunker and Goslin,1998). Briefly, fetuses were removed on embryonic day 18 fromfemale rats anaesthetised with ether and killed by decapitation.Cortices were dissected and placed in Ca2+ and Mg2+ freeHank's balanced salt solution (HBSS) supplemented with 1%Hepes-NaOH (500 mM), 1.82% glucose (30% w/v) andpenicillin/streptomycin (100 U/ml and 100 μg/ml). Cells weredissociated by gentle titration and washed once with Dulbecco'smodified Eagle's medium (D-MEM) without sodium pyruvateand containing high glucose, 2% heat-inactivated fetal bovineserum (FBS), 1% L-glutamine (200 mM) and penicillin/streptomycin (100 U/ml and 100 μg/ml). Cells were thenpelletted at 800 rpm for 3 min, resuspended in plating medium,and plated on poly-D-lysine (5 μg/ml; MW 30,000–70,000)-coated 96-well flat bottom plates (18,000 cells/well) or in 12-mmMillicell®-PCF (36000 cells/insert; Millipore, Vimodrone, Italy).Plating medium consisted of Neurobasal A Medium 1X, 2%B-27 supplement (50X), 1% L-glutamine (200 mM), and pen-icillin/streptomycin (100 U/ml and 100 μg/ml). Cultures weremaintained in a 5% CO2 humidified atmosphere at 37 °C for atleast 8 days, to allow cells sufficient time to adhere to the dishand to form neuronal networks (Bunker and Goslin, 1998).

Principles of good laboratory animal care were followed andanimal experimentation was in compliance with a specific EUdirective (86/609/EEC).

2.3. Analysis of cell viability

To determine the effects of Lioresal® and (RS)-baclofen oncell viability, we carried out the MTTcolorimetric assay. Briefly,after 11 days in culture, cells were treated with Lioresal® or(RS)-baclofen at the indicated concentrations and times. At theend of the incubation, MTT (0.5 mg/ml final concentration) wasadded to each well and the incubation was then continued at37 °C for 4 h. The formazan crystals were solubilised by theaddition of 200 μl of 100% dimethyl sulfoxide. Optical densitywas measured on a multiwell scanning spectrophotometer, usinga test wavelength of 570 nm and a reference wavelength of620 nm.

2.4. Data analysis

Statistical analysis of cell proliferation data was performedusing one-way analysis of variance followed by the post-hocanalysis Dunnett's t test. The differences between the groupswere analysed with Bonferroni's t test. All data are presented asmeans±SEM of at least three separate experiments. Allstatistical analyses were performed using GraphPad Prism4.00 (GraphPad Software, Inc. San Diego, CA, USA).

2.5. SEM and TEM analysis

Specimens were fixed in 1% Karnowsky solution 0.1 M insodium cacodylate buffer (pH 7.4) for 6 h at 4 °C and then

Fig. 1. Effect of treatment of cortical neurons with Lioresal® (Lio) and (RS)-baclofen (Bac) 0.02 and 0.2 mg/ml. Besides untreated cells, physiological solution forintrathecal use (Phy) or HCl 1N was used as control conditions. Neurons were treated for 4 days with the indicated compounds as reported in the figure. Results aremeans±SEM, from at least three independent experiments. Statistical analysis was performed with a post-hoc Dunnett's t test versus untreated cells (control), andBonferroni's t test versus control conditions (n.s., pN0.05).

Table 1Optical density values for the indicated conditions obtained from spectrophotometricreading of cells treated with MTT

Group of cells Optical density P values

24 h 4 days 24 h 4 days

Control 1.058±0.032 0.913±0.034Lioresal 0.02 mg/ml 1.072±0.049 0.872±0.040 ns nsPhysiologicalsolution (1%)

1.040±0.038 0.774±0.065 ns ns

Lioresal 0.2 mg/ml 0.970±0.034 0.789±0.042 ns nsPhysiologicalsolution (10%)

0.982±0.030 0.811±0.047 ns ns

Baclofen 0.02 mg/ml 1.097±0.027 0.917±0.040 ns nsHCl 1N (0.008%) 1.043±0.029 0.800±0.032 ns nsBaclofen 0.2 mg/ml 0.970±0.036 0.889±0.042 ns nsHCl 1N (0.08%) 1.056±0.049 0.830±0.028 ns ns

Values represent means±SEM, from at least three independent experiments.Statistical analysis was performedwith a post-hocBonferroni's t test versus controlcondition (n.s., pN0.05).

35A. Dario et al. / European Journal of Pharmacology 550 (2006) 33–38

preserved in the same buffer. For scanning electron microscopy(SEM) investigations, samples were post-fixed in a solution of1% osmium tetroxide and potassium ferrocyanide 1.25% for2 h, dehydrated in an ascending series of ethanol and criticalpoint dried (Emitech K850; Emitech, Baltimore, MD, USA).They were finally mounted on aluminium stubs, covered with8 nm of gold (Emitech K550 sputter), and examined under aPhilips SEM-FEG XL-30 (FEI, Eindhoven, Netherlands). Fortransmission electron microscopy (TEM), samples were post-fixed in 1% OsO4 for 1 h, washed in 0.1 sodium cacodylatebuffer, dehydrated in an ethanol ascending series andpropylenoxide, and then embedded in Epon 812 mixture. Theembedded specimens were cut with a RMC MTX ultramicro-tome (Boeckeler Instruments Inc., Tucson, Arizona, USA) andthe obtained sections were collected on 150 mesh grids.Sections were overstained with uranile acetate and lead acetate,and examined with a Jeol 1010 EX electron microscope (Jeol,Tokyo, Japan).

3. Results

3.1. Effect of Lioresal® upon neuronal viability

The commercial formulation of Lioresal® consists of aracemic mix of both R- and S-baclofen stereoisomers dissolved

in physiological solution at a maximum concentration of 2 mg/ml. This active compound is usually administered via intrathecalpumps at this concentration. In order to evaluate possible toxiceffects of Lioresal® at 2 mg/ml, the cell culture medium has to bereplaced. Unfortunately, the long-term survival of primaryneurons cultures is dependent on “conditioning” of the culture

Fig. 2. Effect of treatment of cortical neurons with HCl 1N. Cells were treatedfor 4 days with HCl 1N at concentrations reported in the figure. Results are meanO.D. (570nm–620 nm)±SEM from at least three independent experiments andvalues. ⁎⁎, pb0.01 versus untreated cells (control), Dunnett's t test.

Fig. 3. Effect of treatment of cortical neurons with (RS)-baclofen 2 and 4 mg/mlor HCl 1N. A. Cells were treated for 24 h and 4 days with (RS)-baclofen 2 mg/ml.B. Cells were treated for 24 h and 4 days with (RS)-baclofen 4 mg/ml. Results aremeans±SEM, from at least three independent experiments. Statistical analysis wasperformed with a post-hoc Bonferroni's t test versus control conditions (n.s.,pN0.05).

36 A. Dario et al. / European Journal of Pharmacology 550 (2006) 33–38

medium: neurons themselves create the correct environment fortheir survival. A partial substitution of the medium (up to onethird) is however tolerated by the cells (Bunker and Goslin,1998).

Owing to this practical problem, preliminary experimentswere performed using concentrations of Lioresal® of 0.02 and0.2 mg/ml. Addition of Lioresal® to the culture medium for 24 hresulted in no observable alteration of mitochondrial oxidativemetabolism, as determined by the MTT test (Fig. 1 panel Aa; O.D. values, obtained from the spectrophotometric readings, arereported for completeness in Table 1). Although replacement ofthe medium was tolerated, the addition of Lioresal® could causeosmotic stress to neurons: to rule out toxicity due to this effect,control experiments with cells to which physiological solutionsimilar to the Lioresal® was added were performed.

Therapeutic effects of Lioresal® are obtained by treatingpatients for periods as long as weeks or months. To simulatechronic treatment and to better evaluate a possible time depen-dence of the effects of Lioresal®, cortical neurons were incu-bated in Lioresal®-containing medium for a period of 4 days. Asreported in Fig. 1 panel Ba, even after 4 days Lioresal® did notaffect cell viability. The same results were also obtained for thesecond and the third days (data not shown). These findingsindicate that Lioresal®, at the concentrations used, can be con-sidered safe.

Our aim, as previously reported, was however to establishthe safety of this drug at a concentration at least 10 times higherthan that used above. We aimed to use (RS)-baclofen (the activeingredient of Lioresal®) for this purpose. However (RS)-baclofen has a poor water solubility (4 mg/ml) but can besolubilized at a higher concentration (250 mg/ml) in HCl 1N.We therefore decided to try to use this solution in order to testwhether baclofen 2 or 4 mg/ml had toxic effects on neuronsurvival.

First, we re-examined the effect of 0.02 and 0.2 mg/mlconcentrations under this new condition to evaluate possibledifferences with the same concentrations of Lioresal®. HClalone was added to culture medium in order to evaluate itseffects per se.

Fig. 1 (panels Ab and Bb), as well as O.D. values reported onTable 1, shows that neither baclofen nor HCl (at the two con-

centrations used) had an effect compared with the control (non-treated cortical neurons).

3.2. Effect of therapeutic doses of (RS)-baclofen on cell viability

The HCl-solution of (RS)-baclofen allowed us to test theeffect of this drug on cultured cortical neurons at a clinicallyused concentration (2 mg/ml) and to test the possible toxiceffects of the 4 mg/ml concentration.

First, we assessed the effect of HCl per se at the concen-tration needed to prepare the GABAB agonist at a concentrationof 2 and 4 mg/ml. Fig. 2 demonstrates that addition of HCl ledto neuron death both after 24 h and after 4 days. A similarpattern of cell death was also observed on the second and thirddays (data not shown). Thus HCl-treated neurons were used ascontrol.

Incubating cortical neurons with 2 mg/ml (RS)-baclofen didnot significantly change cell viabilitywith respect control (Fig. 3Aand Table 2).

To better evaluate whether treated neurons showed alterationsin network morphology and cell ultrastructure, scanning (SEM)and transmission (TEM) electron microscopy investigations wereperformed. At SEM observation, control neurons appeared togrow homogeneously on the surface of the Millipore scaffold.

Table 2.Optical density values for the indicated conditions obtained from spectrophotometricreading of cells treated with MTT

Group of cells Optical density Pvalues

24 h 4 days

Control 1.058±0.032 0.913±0.034 n.sBaclofen 2 mg/ml 0.368±0.011 0.205±0.009 aHCl 1N (0.8%) 0.392±0.015 0.256±0.018 aBaclofen 4 mg/ml 0.254±0.012 0.091±0.004 aHCl 1N (1.6%) 0.272±0.009 0.207±0.011 a

Values represent means±SEM, from at least three independent experiments.Statistical analysis was performed on the data collected for cells from the 4-daygroup versus those for the 24 h group. (a, pb0.01; n.s., pN0.05; Bonferroni'st test).

37A. Dario et al. / European Journal of Pharmacology 550 (2006) 33–38

The cell bodies (5–6 μm diameters) were stellate in shape withsome microvilla on their surface. The cells had several cyto-plasmic extensions (axons and dendrites) which created a densethree-dimensional network (Fig. 4 panel 1. Scale bar 5 μm). Someaxons terminated on dendrites or cellular soma (inset in panel A ofFig. 4. Scale bar 1 μm). At TEM analysis the cell bodies of thecontrol cortical neurons appeared to be rich in intracellularorganelles, which were well recognisable: the cisternae of thegranular endoplasmic reticulum appeared to be arranged in afairly ordered array; many polysomes occurred free in the cyto-plasmic matrix between the cisternae; mitochondria appearedround, more or less elongated; and some ipodense vesicles werelocalised near the plasma membrane. It was also possible toobserve a dense network of cytoplasmic extensions transversallyor longitudinally sectioned (150 nm in diameter). Neurofilamentsfilling these extensions were well evident and ran parallel to eachother and to the longitudinal axis of the extension. These features

Fig. 4. SEM and TEM images of control and treated neurons. Panel 1. SEM image5 μm); Inset: magnification of an axon terminating on cellular soma (scale bar 1 μmintact organelles can be seen (see text for details; scale bar 300 nm). Panel 3. SEM imacellular shape are similar to those of control samples; some degenerated neurons are viorganelles and extensions appear normal both in dimension and in shape (scale bar

correspond to the normal organisation of nerve fibres (Fig. 4 panel2. Scale bar 300 nm) (Pannese, 1994).

Confirming the MTT data, SEM and TEM analysis ofneurons treated with 2 mg/ml baclofen revealed no differencefrom control neurons (not shown). This behaviour was seenafter treatment for 24 h and 4 days. These data suggest that aconcentration of 2 mg/ml of (RS)-baclofen is safe in terms ofneuronal survival, at least under our experimental conditions.

These encouraging results convinced us to evaluate the effectof 4 mg/ml (RS)-baclofen on neuronal viability. The results ofthese experiments are summarised in Fig. 3B (O.D. values arereported in Table 2). Throughout the 4 days of treatment,baclofen-treated neurons showed no significant differencesversus control cells, indicating that also at this concentration(RS)-baclofen is not toxic to the cells. Although statistical testsshowed that on the fourth day cell survival was not differentbetween the two groups, we investigated the effect of the higherdose of baclofen in greater details by performing an electronmicroscopy analysis (Fig. 4).

The SEM observation of baclofen (4 mg/ml, 4th day)-treatedsamples did not show any alteration in the density of theneurons on the scaffold surface. The cells appeared similar indiameter and in shape to the control neurons. Some degeneratedcell bodies (asterisk in panel 3 of Fig. 4) with irregular surfacewere seen. The three-dimensional network was well developed,similar to that of control neurons (Fig. 4 panel 3, scale bar5 μm). Baclofen-treated cells were not different from controlcells under TEM analysis. In fact, neurons with a cytoplasm richin endoplasmic reticulum, mitochondria, polysomes andvesicles were visible. Also the cytoplasmic extensions wereabundant and well defined. Treated neurons did not showalterations in cell diameter or organisation. The neurofilaments

of control samples showing the neuronal network and the cell bodies (scale bar). Panel 2. TEM image of control samples illustrating the cellular organisation:ge of baclofen (4 mg/ml, 4th day)-treated neurons: the neuronal network and thesible (asterisk; scale bar 5 μm). Panel 4. TEM image of treated cells: cytoplasmic300 nm).

38 A. Dario et al. / European Journal of Pharmacology 550 (2006) 33–38

were evident, packaged in the cytoplasmic extensions parallel tothe major axis (Fig. 4 panel 4, scale bar 300 nm).

Taken together, these findings, although they represent thefirst step in the evaluation of (RS)-baclofen toxicity, show thatthis drug can be considered safe at the concentrations used inour experimental model.

4. Discussion

Lioresal® has been successfully used in clinical trails for thetreatment of spasticity following spinal cord injury (Orsnes et al.,2000). It is administered intrathecally at a concentration of 2 mg/ml. However its possible toxic effects on neuronal cells have notbeen tested under these conditions. Moreover, its activecompound ((RS)-baclofen) is important in the central nervoussystem and diseases or metabolic alterations (Bowery et al.,2002).

The first step in developing future or possible clinical appli-cations of intrathecal baclofen is to study cortical neuron via-bility at different drug concentrations, to identify the safemaximum dosage. Primary cortical cultures represent a strongcellular model with which to perform cell viability tests (as theMTT test used in this work). We first showed that Lioresal®,used at concentrations 10 to 100 times lower than the clinicallyused concentration, was absolutely safe (Fig. 1, Aa and Ba;Table 1) and did not affect in any way the survival of neurons.Also (RS)-baclofen at the same concentrations, althoughdissolved in HCl, did not alter mitochondrial oxidative metab-olism (Fig. 1Ab and Bb; Table 1). This was also true aftertreatment for 4 days. (RS)-Baclofen can therefore be used todetermine the safety or dangerousness of a concentration of atleast 2 mg/ml on neuronal viability.

It is worthwhile to note that neuronal viability is per seinfluenced by days in culture: the number of living neuronsdecreased from 24 h to 4 days of culture (see Fig. 2). As aninternal control, the same reduction was evident also when cellswere treated with baclofen (all concentrations) for 24 h and the4 days.

Furthermore, as shown (Fig. 2 and Table 2), treatment ofcortical neurons with HCl (at a percentage of 0.8% and 1.6%)caused marked cell death. More cells died after 4 days of culturewith baclofen (2 mg/ml and 4 mg/ml) or HCl (0.8% and 1.6%)than after 24 h of culture with the same concentrations (Table 2).There was no significant difference between the effects ofbaclofen and HCl (Fig. 3). Their results therefore suggest that,under our experimental conditions, neuronal survival is affectedby HCl rather than by baclofen.

Our results indicate that, under the described experimentalconditions, baclofen at 2 and 4 mg/ml can be considered safe interms of neuronal survival. Although, it should be rememberedthat we tested the toxic effects of (RS)-baclofen on a residualpopulation (about 30% in the best case), these results arecertainly encouraging for the potential use of Lioresal® in thetreatment of conditions such as pain, cerebral ischemia,Parkinson's disease and peripheral nerve injury.

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