Journal International Medical Research 2009 Berns 680 8

8/11/2019 Journal International Medical Research 2009 Berns 680 8

1/10

http://imr.sagepub.com/ Research

Journal of International Medical

http://imr.sagepub.com/content/37/3/680

The online version of this article can be found at:

DOI: 10.1177/147323000903700311 2009 37: 680Journal of International Medical Research

M Berns, L Seeberg, M Schmidt and T KernerNeurodegeneration in Primary Neuronal Cultures from Rat Embryos

High-dose Propofol Triggers Short-term Neuroprotection and Long-term

Published by:

http://www.sagepublications.com

can be found at:Journal of International Medical Research Additional services and information for

http://imr.sagepub.com/cgi/alertsEmail Alerts:

http://imr.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

What is This?

- May 1, 2009Version of Record>>

by guest on September 7, 2014imr.sagepub.comDownloaded from by guest on September 7, 2014imr.sagepub.comDownloaded from

8/11/2019 Journal International Medical Research 2009 Berns 680 8

2/10

The Journal of International Medical Research2009; 37: 680 688

68 0

High-dose Propofol Triggers Short-term

Neuroprotection and Long-termNeurodegeneration in Primary NeuronalCultures from Rat EmbryosM BERNS1*, L SEEBERG2*, M SCHMIDT 2 AND T KERNER2

1Department of Neonatology, Charit Centre 17 for Gynaecology, Perinatal, Paediatric andAdolescent Medicine with Perinatal Centre and Human Genetics, Campus Virchow-

Klinikum, and 2Department of Anaesthesiology and Operative Intensive Care Medicine,Charit Centre 7 for Anaesthesiology, Operating-Room Management and Intensive Care

Medicine, Campus Virchow-Klinikum and Charit Campus Mitte, Charit-Universittsmedizin Berlin, Berlin, Germany

This study investigated the effects of propofol on primary neuronal culturesfrom rat embryos. Primary corticalneuronal cultures were prepared fromWistar rat embryos (E18). The viability of cells exposed to 0.01, 0.1 or 1 mg/mlpropofol for up to 48 h was assessed usinga methyltetrazolium assay. In order toevaluate the role of g -aminobutyric acid-A(GABA A) receptors, cells were also pre-incubated with the GABA A-receptorantagonists, gabazine and picrotoxin.Propofol at a concentration of 1 mg/mlsignificantly reduced cell viability after 12

h. In contrast, this concentration led to asignificant increase in cell viability at 3and 6 h. The GABA A-receptor antagonistsdid not influence the neurodegenerativeeffect of propofol but abolished itsneuroprotective effect. DNAfragmentation as a marker of apoptosiswas elevated after 24 h propofoltreatment. These results confirm that highdoses of propofol can cause GABA A-receptor triggered neuroprotection and asubsequent time-dependent, but GABA A-independent, neurodegeneration inprimary cortical neurons.

KEY WORDS: P ROPOFOL ; ANAESTHETIC ; NEURODEGENERATION ; NEUROPROTECTION ; CELL VIABILITY;g -AMINOBUTYRIC ACID -A RECEPTOR ; GABAZINE ; PICROTOXIN

IntroductionThere is an indisputable need for generalanaesthesia in newborns and infantsundergoing diagnostic or surgical

interventions. The increasing complexity inthe combination of sedative, anxiolytic and

analgesic drugs used is, however,contradictory to the current state of researchknowledge about the effects of these agents.

The findings of Ikonomidou et al .1 in 2000

confirmed the suspicion that induction of neurodegeneration in the embryonic braincould be triggered via N -methyl- D-aspartate(NMDA)-receptor antagonists and*M Berns and L Seeberg contributed equally to this study.

by guest on September 7, 2014imr.sagepub.comDownloaded from

8/11/2019 Journal International Medical Research 2009 Berns 680 8

3/10

68 1

M Berns, L Seeberg, M Schmidt et al .Effects of propofol on neuronal cells

-aminobutyric acid-A (GABA A)-receptor agonists. 1 Since then, neurodegenerativeeffects have been demonstrated for variousanaesthetic agents, including ketamine,barbiturates, midazolam, isoflurane, nitrousoxide and propofol, 2 5 which are thought toact via GABA A agonism and/or glutamateantagonism. 6 It has become clear that theneurodegenerative effects of anaesthetics aredependent on when they are administered. 7

The strongest effect was observed whenanaesthetics were applied during the braingrowth spurt. 8 During this stage of development, crucial structural alterationsoccur, such as the formation of neuralconnections and dendritic and axonalmaturation, as well as physiologicalapoptosis. This period spans from the thirdtrimester of pregnancy to several years after birth in humans, 5 whereas in rodents itcorresponds to the first three post-natalweeks. 1,5,9

Propofol, an alkyl phenol derivatedissolved in a lipid emulsion, was introducedas an anaesthetic agent in 1977 10 and iswidely used for intravenous induction andmaintenance of a surgical plane inpaediatric anaesthesia. Even though its usefor sedation in critical care has beenrestricted following reports of children dyingfrom myocardial failure after long-term

propofol infusion, 11 its application inchildren older than 2 months for themaintenance of anaesthesia is considered tobe safe. 12 Despite these restrictions, the off-label use of propofol anaesthesia is commoneven in pre-term infants and neonates.

The uncertainty surrounding the safety of the anaesthetics used in neonatalanaesthesia highlights the importance of

further investigations. In view of thecomplexity of validation of anaesthesia for humans in clinical practice, in vitro modelsare useful tools to examine the potential

adverse effects of pharmacological agentsduring brain development.

The aim of this study was to investigatethe effects of propofol on the survival of primary neuronal cells from rat embryosand to elucidate its mechanisms of actionduring the developmental period, includingthe possible role of GABA A receptors.

Materials and methodsPRIMARY CORTICAL NEURONALCULTUREAll procedures were performed according tothe local guidelines for animal researchapproved by the Charit-UniversittsmedizinBerlin. Primary neuronal cultures wereprepared as described elsewhere. 13 Briefly,cerebral cortices from Wistar rat embryos(E18) (Centre for Experimental MedicalResearch, Charit-UniversittsmedizinBerlin) were dissected in serum-freeneurobasal medium with B27 supplement(Gibco-Invitrogen, Karlsruhe, Germany) andthe meninges were removed. 13,14 After trypsinization for 15 min at 37 C intrypsin/ethylenediaminetetra-acetic acid(0.05/0.02%) in phosphate-buffered saline (1mM KH 2PO 4, 155 mM NaCl, 2.96 mMNa 2HPO 47H 2O, pH 7.4; Biochrom, BerlinGermany), the fragments were rinsed twicewith phosphate-buffered saline and once

with dissociation medium (neurobasalmedium with 2% B27 supplement, 1%penicillin and streptomycin and 1% L-glutamine), and then subjected tomechanical dissociation by repeatedaspirations through a fire-polished Pasteur pipette in dissociation medium. The cellsuspension was pelleted by centrifugation(1200 rpm) for 2 min at room temperature

(Minifuge GL; Heraeus Instruments,Fellbach, Germany) and seeded in flat-bottomed wells in neurobasal medium withB27 supplement containing L-glutamine,

by guest on September 7, 2014imr.sagepub.comDownloaded from

8/11/2019 Journal International Medical Research 2009 Berns 680 8

4/10

68 2

M Berns, L Seeberg, M Schmidt et al .Effects of propofol on neuronal cells

penicillin and streptomycin at a density of 3 106 cells/ml. Prior to cell plating, wellswere treated with poly- D-lysine (0.5% inwater; Sigma, St Louis, MO, USA) for 1 h atroom temperature and rinsed twice withwater. Cells were stored in a fully humidifiedincubator at 37 C with 5% CO 2. After 7 daysin vitro, 50% of the medium was removedand replaced with fresh medium.

CELL TREATMENTSAfter a total of 15 days in vitro, cells werethen exposed in three series to 0.01, 0.1 or 1mg/ml propofol for 3, 6, 9, 12, 24 and 48 hor to an equivalent dose of the vehicle(ClinOleic; Baxter, Heidelberg, Germany).Control cells remained untreated. All plateswere placed in the same incubator.

To explore the influence of GABA Areceptors, untreated cell plates were pre-incubated with the GABA A-receptor antagonists gabazine (0.1 mM) andpicrotoxin (0.1 mM) for 30 min. Thesecultures were then exposed to 1 mg/mlpropofol and either gabazine (0.1 mM) or picrotoxin (0.1 mM).

CELL VIABILITY ASSAYThe methyltetrazolium (MTT) assay, basedon reduction of the tetrazolium salt 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetra-

zolium bromide (Sigma) to colouredformazan by active dehydrogenases withinlive mitochondria, was used to determine cellviability. 15 Neuronal cells were plated onto96-well plates coated with poly- D-lysine in100 l aliquots (60 000 cells/well) and treatedas described above. Control cells were kept inneurobasal medium. After 3, 6, 9, 12, 24 and48 h, 10 l MTT at a final concentration of

0.5 mg/ml was added for 2 h. Insolublepurple formazan grains were solubilized byadding 100 l of 10% sodium dodecylsulphate and 0.01 M HCl. Absorbances at

570 nm and the reference wavelength 630nm were measured using a microplate reader (Bio-Rad Laboratories, Munich, Germany).

LIGHT PHASE CONTRAST PHOTOMICROGRAPHSTo visualize the cell damage caused bypropofol (1 mg/ml), representative photo-micrographs were taken of exposed andcontrol cultures using an Olympus digitalcamera (Olympus, Tokyo, Japan) with a 40objective lens.

DETECTION OF CELL DEATHTo quantify apoptosis and necrosis, histone-complexed DNA fragments from thecytoplasm of apoptotic cells or released fromnecrotic cells were detected using an enzyme-linked immunoassay (ELISA) and quantifiedspectrophotometrically. After incubation of thecells with propofol as described above, 20 lcell incubating medium containing DNA fromnecrotic cells that had leaked through themembrane during incubation was transferredto a streptavidin-coated well. The remainingmedium was discarded. To determine theamount of apoptotic nucleosomes, theapoptotic cells were lysed and centrifuged, andan aliquot of the supernatant was transferredto a streptavidin-coated well. The oligosomalDNA was quantified using Cell Death

Detection ELISA PLUS (Roche Diagnostics,Grenzach-Wyhlen, Germany) according to themanufacturers protocol. Results wereexpressed as absorbance of the supernatant of the lysate or the incubating medium of thepropofol-treated cells against that of untreatedcells as controls.

STATISTICAL ANALYSIS

Data were presented as means SEM.Statistical comparisons between the normaldistributed groups were performed usinganalysis of variance and the Bonferroni post-

by guest on September 7, 2014imr.sagepub.comDownloaded from

8/11/2019 Journal International Medical Research 2009 Berns 680 8

5/10

68 3

M Berns, L Seeberg, M Schmidt et al .Effects of propofol on neuronal cells

test or Students t -test (GraphPad Prism 4.03,GraphPad Software, San Diego, CA, USA).All reported P-values were two-tailed; a P-value < 0.05 was considered to indicatestatistical significance.

ResultsCELL VIABILITY AFTER PROPOFOLTREATMENT Cell viability after propofol treatment, givenas the percentage change in cell viabilityrelative to the untreated control group, isshown in Fig. 1. Vehicle-treated cells andcells treated with 0.01 mg/ml propofolshowed no significant differences comparedwith the untreated controls, whereas 0.1mg/ml propofol led to significantly reducedcell viability after 48 h of treatment (29.8 5.3%, P < 0.05). Cell cultures treated with 1mg/ml propofol displayed a significantdecrease in cell viability after only 12 h(17.9 6.9%, P < 0.05), which became moreaccentuated after 24 h (57.3 5.9%,P < 0.001) and 48 h (77.6 4.0%, P < 0.001)compared with untreated controls. In

contrast, short-term exposure to 1 mg/mlpropofol led to significantly higher cellviability (+50.1 12.1% after 3 h, P < 0.01;+22.6 8.5% after 6 h, P < 0.05).

Light phase contrast photographs taken toillustrate the microscopical differences showedmarkedly affected cells after incubation with 1mg/ml propofol for 24 h: control cell showedlong processes with branching (Fig. 2A); cellsincubated with propofol were almostcompletely solved, cell bodies and processeswere nearly lost (Fig. 2B).

CELL VIABILITY AFTER PROPOFOLAND GABA A-RECEPTORANTAGONIST TREATMENTSAfter 3, 6, 9 and 12 h treatment with 1mg/ml propofol and the GABA A-receptor antagonists gabazine and picrotoxin,significant differences in cell viability wereseen compared with that of treated controlcells (treated only with 1 mg/ml propofol)(Fig. 3). The early significant increase in cellviability of propofol-exposed cells wasabrogated by both gabazine (from +50.1 to

FIGURE 1: Cell viability determined by methyltetrazolium (MTT) assay relative tountreated controls in neonatal primary neuronal cultures from rat embryos after 3, 6,9, 12, 24 or 48 h of treatment with 0.01, 0.1 or 1 mg/ml propofol (* P < 0.05; **P 4 mg/kg per day) for 48 h. Eventhough the present study was carried out inneuronal cell culture, the results may be anadditional indicator that propofol should beused with caution for long-term sedation andhigh doses should be avoided in infants.

The advantage of using a primaryneuronal culture is the high survival of plated cells in serum-free B27/neurobasalmedium. 13 In addition, the use of primaryneuronal cultures from rat embryos allowsstudy of the neurodegenerative effects on

developing neurons. However, since theexamined cells are considered to be matureafter 15 days in vitro, further studies shouldbe performed using more immature cells.

This study demonstrated for the first time acombination of time-dependent neuro-protection and neurodegeneration whenpropofol was applied to primary embryoniccultures. Further investigations such asWestern blot analysis of the activation of caspases are needed to identify the underlyingmechanism of neuronal damage anddetermine to what extent establishedanaesthetic procedures may pose a significantrisk to the developing human brain.

AcknowledgementThis work was supported by internaluniversity research grants (Charit-Universittsmedizin Berlin, Germany).

Conflicts of interestThe authors had no conflicts of interest todeclare in relation to this article.

68 7

M Berns, L Seeberg, M Schmidt et al .Effects of propofol on neuronal cells

by guest on September 7, 2014imr.sagepub.comDownloaded from

8/11/2019 Journal International Medical Research 2009 Berns 680 8

10/10

differentiation of CNS precursors in loweredoxygen. J Neurosci 2000; 20: 7377 7383.

11 Parke TJ, Stevens JE, Rice AS, et al : Metabolicacidosis and fatal myocardial failure after

propofol infusion in children: five case reports.BMJ 1992; 305: 613 616.12 AstraZeneca: Product Information: Diprivan .

Wilmington: AstraZeneca, issued April 2001,reviewed August 2001.

13 Brewer GJ: Serum-free B27/neurobasal mediumsupports differentiated growth of neurons fromthe striatum, substantia nigra, septum, cerebralcortex, cerebellum, and dentate gyrus. J Neurosci Res 1995; 42: 674 683.

14 Ruscher K, Freyer D, Karsch M, et al :Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence froman in vitro model. J Neurosci 2002; 22: 10291 10301.

15 Bhrer C, Atzpodien J, Oz S, et al : Fosfomycindoes not reduce cytostatic activity of cis-platinumagainst human osteosarcoma cell lines in vitro.Pediatr Hematol Oncol 1991; 8: 243 249.

16 Kawaguchi M, Furuya H, Patel PM:Neuroprotective effects of anesthetic agents. J Anesth 2005; 19: 150 156.

17 Peters CE, Korcok J, Gelb AW, et al : Anestheticconcentrations of propofol protect againstoxidative stress in primary astrocyte cultures:comparison with hypothermia. Anesthesiology 2001; 94: 313 321.

18 Velly LJ, Guillet BA, Masmejean FM, et al :Neuroprotective effects of propofol in a modelof ischemic cortical cell cultures: role of glutamate and its transporters. Anesthesiology 2003; 99: 368 375.

19 Acquaviva R, Campisi A, Murabito P, et al :Propofol attenuates peroxynitrite-mediatedDNA damage and apoptosis in culturedastrocytes: an alternative protectivemechanism. Anesthesiology 2004; 101: 1363 1371.

20 Mathy-Hartert M, Mouithys-Mickalad A,Kohnen S, et al : Effects of propofol on endothelialcells subjected to a peroxynitrite donor (SIN-1). Anaesthesia 2000; 55: 1066 1071.

21 I to H, Watanabe Y, Isshiki A, et al :Neuroprotective properties of propofol andmidazolam, but not pentobarbital, onneuronal damage induced by forebrainischemia, based on the GABA A receptors. Acta Anaesthesiol Scand 1999; 43: 153 162.

22 Arden N, Betenbaugh MJ: Life and death inmammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 2004; 22:174 180.

23 Arden N, Betenbaugh MJ: Regulating apoptosisin mammalian cell cultures. Cytotechnology 2006; 50: 77 92.

24 Ikonomidou C, Bosch F, Miksa M, et al :Blockade of NMDA receptors and apoptoticneurodegeneration in the developing brain.Science 1999; 283: 70 74.

25 OShea SM, Wong LC, Harrison NL: Propofolincreases agonist efficacy at the GABA Areceptor. Brain Res 2000; 852: 344 348.

26 Spahr-Schopfer I, Vutskits L, Toni N, et al :Differential neurotoxic effects of propofol ondissociated cortical cells and organotypichippocampal cultures. Anesthesiology 2000; 92:1408 1417.

27 Gascon E, Vutskits L, Zhang H, et al : Sequentialactivation of p75 and TrkB is involved indendritic development of subventricular zone-derived neuronal progenitors in vitro. Eur J Neurosci 2005; 21: 69 80.

28 Al-Jahdari WS, Saito S, Nakano T, et al : Propofolinduces growth cone collapse and neuriteretractions in chick explant culture. Can J Anaesth 2006; 53: 1078 1085.

29 Honegger P, Matthieu JM: Selective toxicity of the general anesthetic propofol for GABAergicneurons in rat brain cell cultures. J Neurosci Res 1996; 45: 631 636.

30 Cattano D, Young C, Straiko MM, et al :Subanesthetic doses of propofol induceneuroapoptosis in the infant mouse brain. Anesth Analg 2008; 106: 1712 1714.

31 Engdahl O, Abrahams M, Bjrnsson A, et al .Cerebrospinal fluid concentrations of propofolduring anaesthesia in humans. Br J Anaesth1998; 81: 957 959.

32 Kuan CY, Roth KA, Flavell RA, et al :Mechanisms of programmed cell death in thedeveloping brain. Trends Neurosci 2000; 23: 291 297.

33 Dikranian K, Ishimaru MJ, Tenkova T, et al :Apoptosis in the in vivo mammalian forebrain.Neurobiol Dis 2001; 8: 359 379.

34 Bhutta AT, Anand KJ: Vulnerability of thedeveloping brain. Neuronal mechanisms. ClinPerinatol 2002; 29: 357 372.

35 Bray RJ:Propofol infusion syndrome in children.Paediatr Anaesth 1998; 8: 491 499.

Authors address for correspondence

Dr Monika BernsDepartment of Neonatology, Charit-Universittsmedizin Berlin, Augustenburger Platz 1,

13353 Berlin, Germany.E-mail: monika.berns@charite.de

68 8

M Berns, L Seeberg, M Schmidt et al .Effects of propofol on neuronal cells

by guest on September 7, 2014imr.sagepub.comDownloaded from