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Journal of International Medical
http://imr.sagepub.com/content/37/3/680
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DOI: 10.1177/147323000903700311
2009 37: 680 Journal of International Medical Research
M Berns, L Seeberg, M Schmidt and T Kerner
Neurodegeneration in Primary Neuronal Cultures from Rat Embryos
High-dose Propofol Triggers Short-term Neuroprotection and Long-term

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The Journal of International Medical Research
2009; 37: 680 688
680
High-dose Propofol Triggers Short-term
Neuroprotection and Long-term
Neurodegeneration in Primary Neuronal
Cultures from Rat Embryos
M BERNS
1
*, L SEEBERG
2
*, M SCHMIDT
2
AND T KERNER
2
1
Department of Neonatology, Charit Centre 17 for Gynaecology, Perinatal, Paediatric and
Adolescent Medicine with Perinatal Centre and Human Genetics, Campus Virchow-
Klinikum, and
2
Department 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 cultures
from rat embryos. Primary cortical
neuronal cultures were prepared from
Wistar rat embryos (E18). The viability of
cells exposed to 0.01, 0.1 or 1 mg/ml
propofol for up to 48 h was assessed using
a methyltetrazolium assay. In order to
evaluate the role of g-aminobutyric acid-A
(GABA
A
) receptors, cells were also pre-
incubated with the GABA
A
-receptor
antagonists, gabazine and picrotoxin.
Propofol at a concentration of 1 mg/ml
significantly reduced cell viability after 12
h. In contrast, this concentration led to a
significant increase in cell viability at 3
and 6 h. The GABA
A
-receptor antagonists
did not influence the neurodegenerative
effect of propofol but abolished its
neuroprotective effect. DNA
fragmentation as a marker of apoptosis
was elevated after 24 h propofol
treatment. These results confirm that high
doses of propofol can cause GABA
A
-
receptor triggered neuroprotection and a
subsequent time-dependent, but GABA
A
-
independent, neurodegeneration in
primary cortical neurons.
KEY WORDS: PROPOFOL; ANAESTHETIC; NEURODEGENERATION; NEUROPROTECTION; CELL VIABILITY;
g-AMINOBUTYRIC ACID-A RECEPTOR; GABAZINE; PICROTOXIN
Introduction
There is an indisputable need for general
anaesthesia in newborns and infants
undergoing diagnostic or surgical
interventions. The increasing complexity in
the combination of sedative, anxiolytic and
analgesic drugs used is, however,
contradictory to the current state of research
knowledge 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 brain
could be triggered via N-methyl-D-aspartate
(NMDA)-receptor antagonists and *M Berns and L Seeberg contributed equally to this study.
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681
M Berns, L Seeberg, M Schmidt et al.
Effects of propofol on neuronal cells
-aminobutyric acid-A (GABA
A
)-receptor
agonists.
1
Since then, neurodegenerative
effects have been demonstrated for various
anaesthetic agents, including ketamine,
barbiturates, midazolam, isoflurane, nitrous
oxide and propofol,
2 5
which are thought to
act via GABA
A
agonism and/or glutamate
antagonism.
6
It has become clear that the
neurodegenerative effects of anaesthetics are
dependent on when they are administered.
7
The strongest effect was observed when
anaesthetics were applied during the brain
growth spurt.
8
During this stage of
development, crucial structural alterations
occur, such as the formation of neural
connections and dendritic and axonal
maturation, as well as physiological
apoptosis. This period spans from the third
trimester of pregnancy to several years after
birth in humans,
5
whereas in rodents it
corresponds to the first three post-natal
weeks.
1,5,9
Propofol, an alkyl phenol derivate
dissolved in a lipid emulsion, was introduced
as an anaesthetic agent in 1977
10
and is
widely used for intravenous induction and
maintenance of a surgical plane in
paediatric anaesthesia. Even though its use
for sedation in critical care has been
restricted following reports of children dying
from myocardial failure after long-term
propofol infusion,
11
its application in
children older than 2 months for the
maintenance of anaesthesia is considered to
be safe.
12
Despite these restrictions, the off-
label use of propofol anaesthesia is common
even in pre-term infants and neonates.
The uncertainty surrounding the safety of
the anaesthetics used in neonatal
anaesthesia highlights the importance of
further investigations. In view of the
complexity of validation of anaesthesia for
humans in clinical practice, in vitro models
are useful tools to examine the potential
adverse effects of pharmacological agents
during brain development.
The aim of this study was to investigate
the effects of propofol on the survival of
primary neuronal cells from rat embryos
and to elucidate its mechanisms of action
during the developmental period, including
the possible role of GABA
A
receptors.
Materials and methods
PRIMARY CORTICAL NEURONAL
CULTURE
All procedures were performed according to
the local guidelines for animal research
approved by the Charit-Universittsmedizin
Berlin. Primary neuronal cultures were
prepared as described elsewhere.
13
Briefly,
cerebral cortices from Wistar rat embryos
(E18) (Centre for Experimental Medical
Research, Charit-Universittsmedizin
Berlin) were dissected in serum-free
neurobasal medium with B27 supplement
(Gibco-Invitrogen, Karlsruhe, Germany) and
the meninges were removed.
13,14
After
trypsinization for 15 min at 37 C in
trypsin/ethylenediaminetetra-acetic acid
(0.05/0.02%) in phosphate-buffered saline (1
mM KH
2
PO
4
, 155 mM NaCl, 2.96 mM
Na
2
HPO
4
7H
2
O, pH 7.4; Biochrom, Berlin
Germany), the fragments were rinsed twice
with phosphate-buffered saline and once
with dissociation medium (neurobasal
medium with 2% B27 supplement, 1%
penicillin and streptomycin and 1% L-
glutamine), and then subjected to
mechanical dissociation by repeated
aspirations through a fire-polished Pasteur
pipette in dissociation medium. The cell
suspension 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 with
B27 supplement containing L-glutamine,
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682
M Berns, L Seeberg, M Schmidt et al.
Effects of propofol on neuronal cells
penicillin and streptomycin at a density of
3 10
6
cells/ml. Prior to cell plating, wells
were treated with poly-D-lysine (0.5% in
water; Sigma, St Louis, MO, USA) for 1 h at
room temperature and rinsed twice with
water. Cells were stored in a fully humidified
incubator at 37C with 5% CO
2.
After 7 days
in vitro, 50% of the medium was removed
and replaced with fresh medium.
CELL TREATMENTS
After a total of 15 days in vitro, cells were
then exposed in three series to 0.01, 0.1 or 1
mg/ml propofol for 3, 6, 9, 12, 24 and 48 h
or to an equivalent dose of the vehicle
(ClinOleic; Baxter, Heidelberg, Germany).
Control cells remained untreated. All plates
were placed in the same incubator.
To explore the influence of GABA
A
receptors, untreated cell plates were pre-
incubated with the GABA
A
-receptor
antagonists gabazine (0.1 mM) and
picrotoxin (0.1 mM) for 30 min. These
cultures were then exposed to 1 mg/ml
propofol and either gabazine (0.1 mM) or
picrotoxin (0.1 mM).
CELL VIABILITY ASSAY
The methyltetrazolium (MTT) assay, based
on reduction of the tetrazolium salt 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetra-
zolium bromide (Sigma) to coloured
formazan by active dehydrogenases within
live mitochondria, was used to determine cell
viability.
15
Neuronal cells were plated onto
96-well plates coated with poly-D-lysine in
100 l aliquots (60000 cells/well) and treated
as described above. Control cells were kept in
neurobasal medium. After 3, 6, 9, 12, 24 and
48 h, 10 l MTT at a final concentration of
0.5 mg/ml was added for 2 h. Insoluble
purple formazan grains were solubilized by
adding 100 l of 10% sodium dodecyl
sulphate and 0.01 M HCl. Absorbances at
570 nm and the reference wavelength 630
nm were measured using a microplate reader
(Bio-Rad Laboratories, Munich, Germany).
LIGHT PHASE CONTRAST
PHOTOMICROGRAPHS
To visualize the cell damage caused by
propofol (1 mg/ml), representative photo-
micrographs were taken of exposed and
control cultures using an Olympus digital
camera (Olympus, Tokyo, Japan) with a 40
objective lens.
DETECTION OF CELL DEATH
To quantify apoptosis and necrosis, histone-
complexed DNA fragments from the
cytoplasm of apoptotic cells or released from
necrotic cells were detected using an enzyme-
linked immunoassay (ELISA) and quantified
spectrophotometrically. After incubation of the
cells with propofol as described above, 20 l
cell incubating medium containing DNA from
necrotic cells that had leaked through the
membrane during incubation was transferred
to a streptavidin-coated well. The remaining
medium was discarded. To determine the
amount of apoptotic nucleosomes, the
apoptotic cells were lysed and centrifuged, and
an aliquot of the supernatant was transferred
to a streptavidin-coated well. The oligosomal
DNA was quantified using Cell Death
Detection ELISA
PLUS
(Roche Diagnostics,
Grenzach-Wyhlen, Germany) according to the
manufacturers protocol. Results were
expressed as absorbance of the supernatant of
the lysate or the incubating medium of the
propofol-treated cells against that of untreated
cells as controls.
STATISTICAL ANALYSIS
Data were presented as means SEM.
Statistical comparisons between the normal
distributed groups were performed using
analysis of variance and the Bonferroni post-
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683
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 indicate
statistical significance.
Results
CELL VIABILITY AFTER PROPOFOL
TREATMENT
Cell viability after propofol treatment, given
as the percentage change in cell viability
relative to the untreated control group, is
shown in Fig. 1. Vehicle-treated cells and
cells treated with 0.01 mg/ml propofol
showed no significant differences compared
with the untreated controls, whereas 0.1
mg/ml propofol led to significantly reduced
cell viability after 48 h of treatment (29.8
5.3%, P < 0.05). Cell cultures treated with 1
mg/ml propofol displayed a significant
decrease in cell viability after only 12 h
(17.9 6.9%, P < 0.05), which became more
accentuated 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/ml
propofol led to significantly higher cell
viability (+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 to
illustrate the microscopical differences showed
markedly affected cells after incubation with 1
mg/ml propofol for 24 h: control cell showed
long processes with branching (Fig. 2A); cells
incubated with propofol were almost
completely solved, cell bodies and processes
were nearly lost (Fig. 2B).
CELL VIABILITY AFTER PROPOFOL
AND GABA
A
-RECEPTOR
ANTAGONIST TREATMENTS
After 3, 6, 9 and 12 h treatment with 1
mg/ml propofol and the GABA
A
-receptor
antagonists gabazine and picrotoxin,
significant differences in cell viability were
seen compared with that of treated control
cells (treated only with 1 mg/ml propofol)
(Fig. 3). The early significant increase in cell
viability of propofol-exposed cells was
abrogated by both gabazine (from +50.1 to
FIGURE 1: Cell viability determined by methyltetrazolium (MTT) assay relative to
untreated 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 <
0.01; ***P < 0.001; data are means SEM)
75
3 h
0.01 mg/ml propofol
50
25
0
25
M
T
T
c
l
e
a
v
a
g
e
(
%
c
h
a
n
g
e
v
s
u
n
t
r
e
a
t
e
d
c
o
n
t
r
o
l
)
50
75
100
**
6 h
*
9 h 12 h
*
*
24 h
***
48 h
***
0.1 mg/ml propofol
1 mg/ml propofol
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684
M Berns, L Seeberg, M Schmidt et al.
Effects of propofol on neuronal cells
+3.2% after 3 h, P < 0.01; from +31.8 to
5.6% after 6 h, P < 0.05) and picrotoxin
(from +50.1 to 1.8% after 3 h, P < 0.01; from
+31.8 to 17.8% after 6 h, P < 0.01). In
addition, there was a significant reduction in
cell viability with gabazine after 9 h (from
+9.3 to 28.2%, P < 0.05), but not after 12 h,
and with picrotoxin after 9 and 12 h (from
+9.3 to 37.0% after 9 h, P < 0.01; from 19.9
to 65.8% after 12 h, P < 0.01) compared
with cells treated with 1 mg/ml propofol
alone. There were no significant differences
FIGURE 2: Light phase contrast photographs showing neonatal primary neuronal cell
cultures from rat embryos (A) before and (B) after incubation with 1 mg/ml propofol
for 24 h (scale bars, 10 m)
A B
FIGURE 3: Cell viability determined by methyltetrazolium (MTT) assay relative to
untreated controls in neonatal primary neuronal cultures from rat embryos after 3, 6,
9, 12, 24 or 48 h of treatment with 1 mg/ml propofol alone or with 1 mg/ml propofol
plus the GABA
A
receptor antagonists gabazine (0.1 mM) and picrotoxin (0.1 mM) (*P
< 0.05; **P < 0.01; data are means SEM)
75
3 h
1 mg/ml propofol
50
25
0
25
M
T
T
c
l
e
a
v
a
g
e
(
%
c
h
a
n
g
e
v
s
u
n
t
r
e
a
t
e
d
c
o
n
t
r
o
l
)
50
75
100
**
**
*
**
*
**
**
6 h 9 h 12 h 24 h 48 h
1 mg/ml propofol + gabazine (0.1mM)
1 mg/ml propofol + picrotoxin (0.1mM)
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685
M Berns, L Seeberg, M Schmidt et al.
Effects of propofol on neuronal cells
in cell viability between the three groups
after 24 and 48 h.
EVALUATION OF TYPE OF CELL
DEATH
Evaluation of the type of cell death after 24 h
of treatment with 1 mg/ml propofol using
the Cell Death Detection ELISA
PLUS
system
revealed a statistically significant increase in
the amount of apoptotic nucleosomes (Fig.
4). Absorbance values of the incubation
medium as a marker for necrotic cell death
showed no significant differences compared
with untreated controls.
Discussion
The present study showed that high
concentrations of propofol (1 mg/ml) can
cause an increase in neuronal cell viability
after 3 and 6 h and a decrease from 12 h
onwards. The addition of GABA
A
-receptor
antagonists led to a significant reduction in
cell viability after 3 12 h of propofol
treatment compared with cells incubated with
1 mg/ml propofol alone. The dominating
mechanism of cell death was apoptosis,
particularly in propofol-exposed cells.
The potential neuroprotective effect of
propofol is consistent with many studies
showing that anaesthetics can have
protective effects against damaging factors.
Three different explanations for the
neuroprotective effects of anaesthetics have
been proposed: (i) scavenging of free
radicals, (ii) inhibition of glutamate release
and (iii) potentiation of GABA
A
-mediated
inhibition of synaptic transmission.
16
For
propofol, increased uptake of glutamate,
17,18
antioxidant properties (scavenging of
peroxynitrite)
19,20
and the crucial role of the
GABA
A
receptor
21
have all been
demonstrated. Although most studies have
examined neuroprotective effects after
ischaemic cerebral injury, the effect of
propofol and other agents, such as
barbiturates and volatile anaesthetics, seems
to be mainly directed against excitotoxic
injury.
16
In the present study, it was assumed
that the standardized medium change at the
beginning of each test, which is necessary in
FIGURE 4: Comparison of the type of cell death in neonatal primary neuronal cultures
from rat embryos following Cell Death Detection ELISA
PLUS
and measurement of
absorbance of the supernatant of the lysate or the incubation medium in cells treated
for 24 h with 1 mg/ml propofol compared with untreated control cells (*P < 0.05;
data are means SEM)
250
200
150
A
b
s
o
r
b
a
n
c
e
x
1
0
3
[
A
4
0
5
n
m

A
4
9
0
n
m
]
100
50
Apoptosis
0
*
Necrosis
Control
1 mg/ml propofol
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686
M Berns, L Seeberg, M Schmidt et al.
Effects of propofol on neuronal cells
order to apply the agent (e.g. propofol) to the
culture, caused circumscribed cell death as a
result of the changed chemical composition
of the medium.
22,23
Pre-treatment with the
competitive GABA
A
-receptor antagonist
gabazine and the non-competitive GABA
A
-
receptor antagonist picrotoxin significantly
inhibited the early neuroprotective effects of
propofol and enhanced its later
neurodegenerative effects, suggesting that
GABA
A
receptors have an important role in
propofol-induced neuroprotection.
The neurodegeneration seen after
prolonged exposure to propofol correlates
with previous reports identifying neuronal
death as a consequence of high-dose propofol
anaesthesia in neonatal rodents.
Anaesthetics, sedatives and anti-epileptics
that act via NMDA-receptor antagonism
and/or GABA
A
-receptor agonism (such as
ketamine, barbiturates, midazolam,
isoflurane, nitrous oxide and propofol) have
been shown to affect brain development in
neonatal rodents.
2,4,5,7,8,24
Accentuated
GABA
A
-receptor agonism and a more
distinctive NMDA-receptor antagonism have
been described as the main mechanisms of
action for propofol.
6,25
Propofol-induced
neurodegenerative effects, which also showed
dose dependence, manifested as persistent
behavioural deficits and a potentiation of
neuronal cell death in studies performed in
neonatal mice.
5
Clinically relevant
concentrations of propofol caused a dose-
dependent loss of GABAergic neurons and
impairment of dendritic arbour development
in primary cultures from newborn rat cerebral
cortex.
26,27
In a study with chick explant
cultures, supraclinical concentrations of
propofol induced neurite retractions and
growth cone collapse.
28
In addition, several
studies have demonstrated much more severe
neurodegeneration triggered by combinations
of anaesthetics, including thiopental plus
propofol, ketamine plus propofol, midazolam
plus ketamine, and diazepam plus
phenobarbital.
2,5,8
GABAergic neurons in rat
brain cell co-cultures have been shown to
display vulnerability to propofol
administration, in contrast to glial cells.
29
High-dose propofol or the combination of
ketamine and propofol was shown to
increase apoptosis measured with Fluoro-
Jade staining in mice brains.
5
In the present
study, the results of the Cell Death Detection
ELISA
PLUS
system argue for the dominance of
an apoptotic mechanism and are, therefore,
consistent with other studies.
5,30
An assumed
GABA potentiation, which would lead to
excessive neuronal inhibition and neuronal
redundancy resulting in apoptosis, is unlikely
in the light of the GABA
A
-receptor
independence of the neurodegenerative effect
of propofol shown in the present study. The
design of the present study does not allow
conclusions concerning the mechanism of
cell death; further investigations are needed
to find out whether low doses of propofol lead
to apoptosis and high doses to necrotic death,
depending on exposure time.
The propofol concentrations used in the
present study are very high compared with
clinical levels and there is currently no clear
evidence of a concentration limit for the
effects seen. After anaesthesia with propofol
at a dose of 2 mg/kg body weight and a
maintenance dosage of 8 mg/kg body weight
per h, a plasma concentration of 2.24 g/ml
after 5 min and a cerebrospinal fluid
concentration of 35.5 ng/ml after 15 30
min were measured in human adults.
31
It is
important to take into account the increased
vulnerability of the immature brain to
neuronal apoptosis or to neurodegenerative
mechanisms caused by natural
reorganization,
32,33
various metabolic events
(such as hypoxia, hypoglycaemia or
ischaemia) or exposure to anaesthetic
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This study demonstrated for the first time a
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cultures. Further investigations such as
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caspases are needed to identify the underlying
mechanism of neuronal damage and
determine to what extent established
anaesthetic procedures may pose a significant
risk to the developing human brain.
Acknowledgement
This work was supported by internal
university research grants (Charit-
Universittsmedizin Berlin, Germany).
Conflicts of interest
The authors had no conflicts of interest to
declare in relation to this article.
687
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Effects of propofol on neuronal cells
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Authors address for correspondence
Dr Monika Berns
Department of Neonatology, Charit-Universittsmedizin Berlin, Augustenburger Platz 1,
13353 Berlin, Germany.
E-mail: monika.berns@charite.de
688
M Berns, L Seeberg, M Schmidt et al.
Effects of propofol on neuronal cells
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