Neuroblastoma Drug Effects
Cells for Testing Neuroprotective
BARBARA PERUCHE AND JOSEF KRIEGLSTEIN
An attempt effects.
was made to use neuroblastoma
To achieve
blastoma containing Drugs
fresh
were
nutrient
medium.
of glucose.
available
damage,
than
naftidrofuryl,
were
ing with
under
induced cyanide
period
to recover
(1 mmol/L) the cyanide-
cells were
for another
to hypoxia
phosphates
drug
in neuro-
until
and culture
addi-
7 days.
24 hr after
protein
development
conof cell
and viability. and phenytoin 300 (*mol/L,
revealed protected
similar experimental drug
that neuroblastoma
neurotoxic
chlorpromazine,
and flunarizine
neuroprotective
suggested
this hypoxic
for the posthypoxic
damage. These results were comparable of neurons
of sodium
30 min prior
of high-energy
neuroprotective
was
after 6 hr by replacing
allowed
as representatives
barbiturates higher
During
They
hypoxia
by addition
to the cells from
determined cell activity
While
cells for testing
cytotoxic
and was terminated
Cell concentration
tent were
tine,
medium
deprived
hypoxia.
doses
damage,
cells after 10 days in culture
to the culture tionally
cellular
effects
effects
dizocilpine,
conditions. obtained
cells are suitable
from
In addition, from
applied
in
ketazo-
cells against hypoxic
neuroblastoma
to those obtained
when
ketamine, primary
cultures
they were
in keep-
in vivo experiments.
for testing
neuroprotective
It is drug
effects.
Key Words: Calcium
Cell
culture;
antagonists;
pressants;
Neuroblastoma
N-methyl-D-aspartate
cells;
Cytotoxic
(NMDA)
hypoxia;
antagonists;
Cyanide; Central
de-
Nootropics
INTRODUCTION Several in vivo models have been developed to investigate the consequences of ischemic and hypoxic damage of brain tissue (Krieglstein et al., 1972; Pulsinelli and Brierley, 1979; Tamura, et al., 1981; Kirino, 1982; Smith et al., 1984). To reduce the complexity of an in vivo model cultured neurons were used. Recently, an in vitro model has been developed in our laboratory for screening neuroprotective drug effects under hypoxic conditions by using neurons of chick embryo cerebral hemispheres as a primary cell culture system (Krieglstein et al., 1988). Cytotoxic hypoxia was induced by addition of sodium cyanide to the culture medium and several drugs revealed neuroprotection by preserving the neuronal ATP level, cell mass or viaFrom the lnstitut ftir Pharmakologie und Toxikologie, Philipps-Universitat Marburg, Ketzerbach 63, 3550 Marburg/Lahn, Germany. Address reprint request to: Professor J. Krieglstein, lnstitut fijr Pharmakologie und Toxikologie, Philipps-Universittit Marburg, Ketzerbach 63, 3550 Marburg/Lahn, Germany. Received December 3, 1990; revised and accepted March 15, 1991.
139 Journal of Pharmacological
Methods
8 1991 Elsevier Science Publishing
26, 139-148 W31) Co., Inc., 655 Avenue of the Americas, New York, NY 10010
140
6. Peruche and J. Krieglstein bility. The model
proved
of drugs (Krieglstein or not comparable For instance, their
character
(Breakfield,
for weeks,
the
protective
effects
with transformed
neuro-2a)
1976;
are no longer
the hypoxic of several
drugs
are experi-
they can be kept
However,
for limited
in order
they
of neurons,
needed.
conditions
cells arose.
to have preserved
et al., 1969);
cultures
using this cell
cell damage
to compare
effects
of whether
neuronal
are known
Nelson
than primary
and animals
line we had to redefine
the neuroprotective
et al., 1990). Now the question
cells (clone
much easier to handle
in culture
to demonstrate
results could be achieved
neuroblastoma
neuronal
mentally
to be suitable
et al., 1988; Peruche
them
and study
with
previous
results. MATERIALS
AND
METHODS
Materials All experiments
were
blastoma,
clone
American
Type Culture
and were
cultivated
carried
neuro-2a
out on cultured
(N2a,
Collection
in Falcon
tumor
passages 168-178). (ATCC,
culture
cells of type C-1300
The cells were
Flow Laboratories
Ltd., Irvine,
flasks (25 and 75 cm’;
neuro-
purchased
Serolab,
from
Scotland)
Aidenbach,
Germany). The following auxiliary materials were purchased from Gibco (Paisley, Scotland) : Dulbecco’s modified Eagle medium (DMEM) with sodium pyruvate and 1 g/L glucose,
Dulbecco’s
icillin/streptomycin tamine Puck’s
Eagle medium
(10000 units/ml
“special”
(without
and 10000 kg/mL,
glucose),
respectively),
penL-glu-
solution 200 mmol/L, nonessential amino acids, trypsine-EDTA solution in salt solution A (PBS). Fetal calf serum was supplied by Biochrom (Berlin,
Germany), albumin
modified
solution
sodium from
cyanide
from
Behringwerke
Merck
(Marburg,
(Darmstadt, Germany).
Germany) All other
and bovine
chemicals
serum
were
of re-
agent grade. chlorpromazine The following drugs were tested: Bayer, Leverkusen, Germany), dizocilpine maleate Harlow,
UK),
flunarizine
dihydrochloride
ketamine hydrochloride (Ketanesta, methanesulfonate (Sterling-Winthrop,
hydrochloride (MK-801,
(Sibelium@,
Parke-Davis,
Janssen,
Berlin,
Neu-lsenburg,
MSD,
(Megaphena, Terlings
Neuss,
Germany),
Germany),
Park
Germany), ketazocine
methohexital
so-
dium
(Brevimytal”, Lilly, Homburg, Germany), naftidrofuryl hydrogenoxalate (Duphenytoin sodium (Phenhydans, Desitin, Hamsodrile, Lipha, Essen, Germany), UCB Chemie, Kerpen, Germany), pyritinol burg, Germany), piracetam (NootropE, hydrochloride
(Encephabol@,
iurn@, Thiemann, Cultivation
Waltrop,
Conditions
Merck,
Darmstadt,
Germany),
vinpocetine
(Eusen-
Germany). of the Neuroblastoma
Cells
The cells were grown in 75 cm2 Falcon culture flasks. They were subcultured every 10 days, after reaching confluency. For hypoxic experiments, the cells were subcultured in 25 cm2 culture flasks. After trypsinization the cells were suspended in 20 mL culture medium and counted using a hemocytometer chamber. One mL (34 x IO5 cells) of this suspension was seeded into each 25 cm2 culture flask containing 5 mL of culture medium. As nutrient medium we used Dulbecco’s modified Eagle
Testing for Neuroprotective medium
with
2 mmol/L
sodium
glutamine,
pyruvate
and 1 g/L glucose
1% nonessential
amino
fortified
Drugs
by 10% fetal calf serum,
acids, and penicillin/streptomycin
(100
units/ml and 100 pg/mL, respectively). The cultures were kept in an incubator (Heraeus, Hanau, Germany) at 37°C in a humidified atmosphere of 95% air and 5% COZ. Cytotoxic Thirty replaced
Hypoxia
and Recovery
10 days of subculture
by adding incubation
a freshly prepared medium
6 hr the cyanide-containing
anide-free recover
Cells
min before hypoxia was started nutrient medium was aspirated off and by DMEM “special” (without glucose). Cytotoxic hypoxia was induced after
to the glucose-free After
of the Neuroblastoma
nutrient
medium
medium
(DMEM
stock solution
of sodium
was removed
and replaced
with 1 g/L glucose).
cyanide
of 1 mmol/L.
to give a final concentration
by fresh cy-
The cells were
allowed
to
for 7 days.
Application
of Drugs
Thirty min before glucose-free after hypoxia.
Control
Determination
the freshly
medium. cultures
Drugs
prepared were
drug solutions present
was stopped
Phosphates
and Protein
and metabolites
and
Passonneau
(1972)
using
the
until
volumes
to the 24 hr
of solvent.
Content
extracted
et al. (1988). ATP, ADP and AMP were measured
to Lowry
were added
in the medium
were treated with corresponding
of High-Energy
Cell metabolism Krieglstein
hypoxia
incubation
as already
described
enzymatically
Aminco-Bowman
by
according
fluorimeter
(Silver
Springs, MD). Energy charge (EC) was determined according to Atkinson (19681, i.e., EC = (ATP + 0.5 ADP)/(ATP + ADP + AMP). The protein content of the cultures
was determined
a protein
by the Biuret method.
Bovine serum albumin
was used as
standard.
Statistics Statistical
comparison
between
the drug-treated
sponding control group was performed by Duncan’s test (Sachs, 1978).
hypoxic
by one-way
groups
and the corre-
analysis of variance
followed
RESULTS Ten days after subcultivation was induced.
Transformed
a confluent
neuronal
cell sheet
had developed
cells are able to cover their
and hypoxia
energy
require-
ments under hypoxic/anoxic conditions by an increased anaerobic glycolysis provided that glucose supply is sufficient (Pauwels et al., 1985). Thus, to induce a deleterious energy failure in the cells as a basis for testing neuroprotective drug effects, it was necessary to combine hypoxia (incubation with NaCN) with glucose deprivation. Central depressant drugs like methohexital and phenytoin showed neurotoxic effects in doses higher than 300 kmol/L. They induced both a marked decrease in the ATP level and the energy charge of the neural cells as well as a loss of protein content of the cultures (Table 1).
141
142
B. Peruche and J. Krieglstein TABLE 1
Neuroprotective
Effect of Drugs Against Hypoxic Damage ATP
DRUG DRUG
CONC.
(kMOL/L)
NACN
CONC.
(MM~L~L)
(NMOL/MC
PROTEIN
ENERGY
PROTEIN)
(MC/FLASK)
CHARGE
Chlorpromazine
0 0.1 1 10 10 0
1 1 1 1 0 0
8.18 t 15.26 + 14.96 t 6.98 t 24.12 c 22.34 +
2.60 2.57** 3.22** 1.45 3.28 1.44
1.50 f 1.77 k 1.69 f 1.69 k 2.86 k 2.78 k
0.06 0.08** 0.19* O.fO* 0.78 0.29
0.87 0.85 0.85 0.87 0.97 0.96
r + + r c c
0.05 0.03 0.06 0.04 0.01 0.01
Dizocilpine
0 0.1 1 IO IO 0
1 1 1 1 0 0
13.19 2 15.29 * 16.09 2 16.55 k 27.82 f 27.50 2
1.60 1.10** l.OO** 0.48** 1.59 1.00
1.55 2 1.73 f 1.78 2 1.79 * 3.58 k 3.76 2
0.08 0.09* 0.13** 0.11** 0.42 0.07
0.91 0.92 0.93 0.94 0.98 0.97
c + r * ? 2
0.03 0.04 0.03 0.02 0.01 0.01
Flunarizine
0 0.1 1 10 IO 0
1 1 1 1 0 0
11.26 f 9.99 k 11.90 * 10.93 f 28.37 + 28.16 c
1.08 1.52 1.09 0.44 0.64 2.17
1.62 2 0.01 1.77 f 0.05' 1.85 IT 0.03"* 1.87 f 0.08** 2.81 k 0.28 2.79 c 0.17
0.85 0.84 0.85 0.85 0.97 0.95
+ 5 + k 2 +
0.01 0.03 0.05 0.02 0.02 0.01
1.55 1.42** 4.63" 0.93** 2.32 2.25
1.54 c 2.62 t 2.52 2 2.34 t 3.10 k 3.18 2
0.74 + 0.98 k 0.98 k 0.99 ? I?0 0.99 f
Ketamine
0 1 IO 100 100 0
1 1 1 1 0 0
10.11 20.05 21.04 20.00 30.79 30.26
Ketazocine
0 1 IO 100 100 0
1 1 1 1 0 0
16.15 ZL 1.37 20.09 k 1.52** 21.47 2 1.72** 23.05 f 1.89** 27.13 t 2.40 26.73 + 1.62
1.69 k 0.08 2.02 2 0.34** 2.04 2 0.28** 2.11 f 0.28** 3.10 IT 0.19 2.77 k 0.21
0.80 0.92 0.89 0.93 0.93 0.93
? 0.02 -c 0.01** " O.Ol** 2 0.03 2 0.02 k 0.02
0 100 300 1000 1000 0
1 1 1 1 0 0
17.01 + 0.86 16.74 2 0.94 16.70 i- 1.89 2.23 2 2.27** 30.68 2 1.10 28.65 k 2.58
1.94 f 0.14 1.79 If-0.07 1.71 + 0.10 0.32 + 0.36" 3.04 * 0.14 2.97 k 0.19
0.95 0.95 0.94 0.37 0.96 0.97
? i k i k k
1 1 1 1 0 0
18.54 k 20.25 k 18.33 2 19.80 2 27.01 k 27.23 +
0.89 1.23 1.15 1.64 1.59 1.34
1.19 I! 0.06 1.48 k O.lO** 1.54 k 0.11** 1.49 2 0.09** 2.77 2 0.26 2.87 k 0.24
0.88 0.92 0.93 0.92 0.94 0.94
k 0.02 k 0.0-l k 0.03 2 0.02 ? 0.02 -c 0.01
1 1 1 1 0 0
12.85 + 12.53 k 12.71 k 8.71 2 20.90 k 27.10 2
2.34 1.20 2.02 1.71** 1.73 2.10
1.53 2 0.09 1.59 ?I 0.06 1.53 k 0.10 0.75 " 0.15** 2.59 * 0.04 2.64 f 0.04
0.86 0.89 0.84 0.58 0.92 0.95
* 0.04 -c 0.05 * 0.05 k 0.04** k 0.02 -c 0.01
Methohexital
Naftidrofuryl
Phenytoin
0 0.1 1 IO IO 0 0 IO 100 1000 1000 0
k 2 k 2 k 2
0.17 0.19** 0.16** 0.38** 0.12 0.16
0.05 0.01" O.Ol** 0.04** 0.01
0.01 0.01 0.03 0.41" 0.01 0.0-I
Testing for Neuroprotective TABLE 1
Drugs
(continued) ATP DRUG CONC.
DRUG
NACN
(,.LMOL/L)
2 1.37
1.40
*
0.29
0.88
?
0.01
13.12
k 1.56
1.66
*
0.15
0.87
?
0.03
1000
14.14
2 2.62
1.44
2
0.31
0.82
"
0.11
10000
13.93
? 2.39
1.70
"
0.18
0.85
2
0.09
0
26.97
t
1.90
2.74
2
0.12
0.94
-+ 0.01
0
27.41
2 2.12
2.58
?
0.21
0.90
2
0.04
10.58
t
0.45
1.61
2
0.05
0.73
*
0.02
10.31
5 0.42
1.54
+
0.08
0.73
2
0.02
100
11.05
k 0.73
1.57
+
0.08
0.71
?
0.06
1000
12.53
t
1.69
1.55
?
0.08
0.73
"
0.06
0 0
1
IO
1000
0
29.44
2 1.40
2.92
+
0.20
0.90
?z 0.01
0
0
29.70
t
2.01
2.79
L
0.11
0.92
2
0.02
0
1
16.22
5 2.17
1.72
?
0.40
0.94
*
0.03
13.55
? 1.96
1.88
?
0.26
0.93
*
0.02
10
12.02
2 1.61
1.83
?
0.34
0.91
*
0.03
100
13.69
-+ 3.11
1.47
*
0.43
0.89
"
0.05
Vinpocetine
Neuroblastoma
100
0
30.88
2 0.63
2.87
?
0.26
0.95
2
0.01
0
0
27.86
k 3.85
2.88
?
0.15
0.97
2
0.01
ceils were
incubated
Recovery
period
for 6 hr with NaCN in glucose-containing
to the cells from 30 min prior
experiments. way analysis
For the statistical of variance
in all applied
hypoxic
as was the protein tures. This could
(NMDA)
content
revealed
and untreated
hypoxic
dizocilpine,
compared
agonist. cells with
as indicated
were
5 SD of six cultures
ketamine
one-
and chlor-
neuroprotective
with
hypoxic
of hypoxic
nootropics
their posthypoxic
after 10
Drugs
** P < 0.01.
significant
after incubation
naftidrofuryl induced a significant increase calcium antagonist flunarizine significantly cell cultures
medium
lasted 7 days.
are given as means
* P < 0.05;
antagonists
of the cultures
also be observed
did not ameliorate
of the neural
Values
drug-treated
applied:
in glucose-free
medium
The ATP level of the cells was significantly
with ketazocine, a kappa-opioid Treatment of neuroblastoma vinpocetine
between
test were
concentrations
cell damage.
(1 mmol/L) nutrient
until 24 hr after hypoxia.
comparison
and Duncan’s
The N-methyl-D-aspartate promazine
ENERGY CHARGE
14.28
Pyritinol
days of cultivation.
PROTEIN (MC/FLASK)
0
10000
against
PROTEIN)
(MM~L/L)
100
Piracetam
available
(NM~L/M~
CONC
nontreated
neuroblastoma
like piracetam,
recovery,
effects elevated,
whereas
pyritinol incubation
culcells and with
in protein content of the cultures. The ameliorated posthypoxic development
by an increased
protein
content.
DISCUSSION In the present study we tried to establish a model of a hypoxic neural cell line for testing neuroprotective drug effects comparable to the above mentioned model of primary neural cell cultures deriving from chick embryo cerebral hemispheres and Krieglstein, 1989; Peruche et al., 1990). (Krieglstein et al., 1988; Ahlemeyer Neuroblastoma
cells (clone
neuro-2a)
were
isolated
from a mouse
tumor
and have
143
144
B. Peruche and J. Krieglstein been cultured
in vitro since 1967 (Augusti-Tocco
primary cell culture system, manageable in vitro model. Despite
and Sato, 1969). In contrast
the cell line in general
their transformation
represents
these cells are able to preserve
a vigorous
to the
and more
their neural character
(Nelson et al., 1969; Breakfield, 1976; Willow et al., 1983), an indispensable requirement for the comparison of the results obtained in the present study with those obtained cultures
under
(Krieglstein
similar
experimental
conditions
et al.., 1988; Ahlemeyer
by using primary
and Krieglstein,
1989;
neural
Peruche
cell
et al.,
1990). The question
arose as to whether
against hypoxia and whether in a similar known
manner
e.g.,
glycolysis.
(Pauwels
we defined
Incubation
of the cells with
recovery in nutrient about 50% of control (120 min)
recovery
complete
cell death
The influence the neural
a marked
or neurons
Pasteur-effect
requirements
as an adaptation experimental
NaCN
(1 mmol/L)
a rapid breakdown
sensitivity
dep-
anaerobic
as long as glucose with glucose
for neuroblastoma
in glucose-free
of energy
It is well
under oxygen
in combination conditions
by drugs
in vivo.
by an increased
to hypoxia
et al., 1985). Using NaCN
ischemia-like
of 6 hr induced
cells reflect a similar against hypoxic damage
cell cultures
cells exhibit
This can be interpreted
deprivation duration
neural
the cells cover their energy
supply is sufficient cells.
as primary
that neuroblastoma
rivation,
neuroblastoma
they can be protected
metabolism.
medium
for a
After 7 days of
medium a restoration of cellular activity and cell growth to was measurable (Figure I). Following shorter hypoxic periods
was accomplished
within
2 days while
12 hr of hypoxia
caused
(data not shown).
of various
drugs on the posthypoxic
cells was determined
by measurement
Posthypoxic
permd
recovery
and development
of the high-energy
of
phosphate
Iweeks)
FIGURE 1. Development of neuronal damage after 6 hr of cytotoxic hypoxia and a recovery period of 2 weeks. After 10 days in culture neuroblastoma cells were incubated with 1 mmol NaCN/L in glucose-free medium for 6 hr. Development of hypoxia-induced neuronal damage was determined during a posthypoxic recovery period of 2 wk. Protein content of the cultures was measured as a reliable indicator of cell growth and viability according to the Biuret method. Data are given as means +SD of six experiments. For the statistical comparison different from the correone-way analysis of variance and Duncan’s test were applied: “P < 0.01, different from the protein content measured imsponding normoxic control: mediately after hypoxia: hP < 0.01.
Testing for Neuroprotective concentration
of the cells and protein
content
of the cultures.
Drugs
Drugs were available
to the cells from 30 min before until 24 hr after hypoxia. It is assumed that oxygen deficiency induces a massive influx of calcium as well as an increased release of excitatory
amino acids both of which
of pathologically Wieloch,
disturbed
1985; Goldberg
induce
metabolic
neural cell death at the end of a cascade
reactions
in vitro and in vivo (e.g.,
et al., 1987). Therefore
we tested
Siesjo and
drugs that are believed
to interrupt these deleterious events, e.g., centrally depressant drugs, NMDA antagonists and calcium antagonists, as well as substances belonging to the group of the so-called
nootropics.
Barbiturates in therapeutic dosages are known to lower energy the cells and thus protect them against hypoxia-induced depletion and subsequent effects
cell death.
in doses higher
by Ahlemeyer
and Krieglstein
experimental
hypoxic
strated chronic
In the present
than 300 pmol/L,
study methohexital
which
is in keeping
(1989) using primary
conditions.
revealed with
neural cell cultures
Roth-Schechter
and Mandel
neurotoxic
results described under
similar
(1977) also demon-
selective neurotoxic effects of pentobarbital (IO pmol/L-I mmol/L) after incubation of mixed cultures of neurons and glia. The differences in the
sensitivity
of these cells to pentobarbital
metabolism. Long-term uncoupling
Neural
cells are more
incubation
with
of oxidative
are explained
sensitive
pentobarbital
in doses
phosphorylation
by differences
to oxygen
et al. (1983).
These
deleterious
effects
deficiency
higher
and thereby
in their energy than glial cells.
than 10 Fmol/L
causes cell damage.
rotoxicity of phenytoin, its influence on ion homeostasis and calcium permeability of the neural cell membrane Swaiman
requirements of of energy stores
induces The neu-
and especially on sodium have been described by
of the drug could
be responsible
for the posthypoxic neural cell damage observed in the present study. Dizocilpine, ketamine and chlorpromazine were able to inhibit hypoxia-induced depletion
TABLE 2
of energy
stores of the cells and protein
loss of the cultures
in all applied
Comparison of Drug Effects In Vitro and In Vivo
Chlorpromazine Dizocilpine Flunarizine Ketamine Ketazocine Methohexital Naftidrofuryl Phenytoin Piracetam Pyritinol Vinpocetine
+ + + + + _
+
+
+ + + + -
+ + -
+ -
+ _ -
+ -
+ -
+ -
+ + +
(Leander, 1989) (Seif el Nasr et al., 1989) (Krieglstein et al., 1989a) (Seif el Nasr et al., 1989) (Tang, 1985) (Karkoutly, 1990) (Krieglstein et al., 198913) (Karkoutly, 1990) (Schmidt et al., 1989) (Schmidt et al., 1989) (Sauer et al., 1988)
+ = neuroprotective effect; - = no effect demonstrable * the results obtained with primary neuronal cell cultures are described by Ahlemeyer and Krieglstein (1989)
145
146
B. Peruche and J. Krieglstein concentrations, tures
which
(Ahlemeyer
promazine
coincides
with results obtained
and Krieglstein,
revealed
1989).
neuroprotective
brain ischemia
of the rat by reducing
hippocampus
formation
of action
the blockade
discussed
(Kemp
itive NMDA
action in histological
(Krieglstein,
1989;
of the calcium
antagonists
binding
receptor
from primary
ketamine,
neural cell damage operated
cell cul-
and chlor-
studies in vivo after fore-
in the selectively 1989).
vulnerable
As a mechanism
by the NMDA
and dizocilpine
are known
to the PCP recognition
site, which
and to the NMDA-gated
neural
dizocilpine
Rod and Auer,
channel
et al., 1987). Ketamine
linked to the NMDA
Except
Ca*+ channel.
receptor
is
as noncompetis allosterically Ketamine
failed
to protect neurons against ischemic damage in vivo (Jensen and Auer, 1988) probably because of its short plasma half life (IO min in rats) (Marietta et al., 1975). Chlorpromazine
is also believed
to interact
with the NMDA-receptor-complex
binding site (Leander, 1989). The in vitro application of ketazocine, protective
effects
in the neural
after hypoxia
(Ahlemeyer
demonstrated
in other
studies.
lwama
a kappa-opioid
cell line as well
and Krieglstein, in vitro
agonist,
as in primary
at the Zn*+ induced
neural
1989). This neuroprotection
(Iwama
et al., 1987) as well
et al. (1987) suggest this substance
neuro-
cell cultures has also been
as in vivo (Tang,
influences
a subtype
1985)
of the so-
dium channel and thus an inhibition of neural activity. As for neuroblastoma electrical membrane activity has been shown (Willow et al., 19831, this could possible
explanation
for the amelioration
the present study. The calcium antagonist effect
also observed
system. This compound by interaction
with
flunarizine
by Ahlemeyer
inhibited to inhibit
binding
prevent consecutive cell death. Nootropics like pyritinol, piracetam tective
effects
in the present
state of the cells obtained
protein
and Krieglstein
is supposed
intracellular
of energy
loss of the cultures
(1989) in a primary
the increase
in cytosolic
sites (van Zwieten,
and vinpocetine
study. This coincides
cells be a in
used, an
cell culture Ca*+
levels
1986) and thus
did not reveal
may
neuropro-
with the results of the long-term
study with primary cultures of neurons described by Ahlemeyer and Krieglstein (1989), except pyritinol, which inhibited protein loss in the primary neural cell culture system. The disagreement not be explained
because
between
results obtained
the mechanism
with pyritinol
of the neuroprotective
in vitro can-
action
for this
drug is unknown. While the vincamine derivative vinpocetine could reduce the development of neural damage after forebrain ischemia in the rat (Sauer et al., 1988), the substance
failed
vitro systems,
probably
to reveal because
neuroprotective of an indirect
effects
in the abovementioned
adenosine
agonistic
effect
in
(Rischke
et al., 1989). Naftidrofuryl is known to inhibit neural damage in vitro (Ahlemeyer and Krieglstein, 1989) and in vivo (Krieglstein et al., 1989b). Protein loss of the neuroblastoma cell culture used in the present study was also inhibited by naftidrofuryl. For this drug action (Fujikara In conclusion,
antiserotonergic et al., 1989). neuroblastoma
effects
are suggested
cells were
as a possible
demonstrated
mechanism
to be suitable
of
for testing
neuroprotective drug effects. The results obtained with this cell line were comparable to those obtained from primary cultures of neurons and even from in vivo
Testing for Neuroprotective
Drugs
models of cerebral ischemia (Table 2). Moreover, neuroblastoma cells seem to be advantageous because they are vigorous and more manageable than primary cultures of neurons. This work was supported by a grant from the Bundesminister fur Forschung und Technologie (Bonn). The authors are indebted to Ms. R. Seidel and Ms. S. Engel for their skillful technical assistance.
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Breakfield X0 (1976) Neurotransmitter metabolism in murine neuroblastoma cells. Life Sci 18:167178. Fujikara H, Kato H, Nakano S, Kogure K (1989) A serotonin SZ antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil. Brain Res 494:387-390. Goldberg MP, Weiss JH, Pham PC, Choi DW (1987) N-methyl-D-aspartate receptors mediate hypoxic neuronal injury in cortical culture. / Pharmacol Exp Ther 243: 784-791. lwama T, lshihara K, Takagi H, Satoh M (1987) Possible mechanism involved in the inhibitory action of U-50,488 E, a kappa-opioid agonist, on guinea pig hippocampal CA3 pyramidal neurons in vitro. / Pharmacobio Dyn 10:56&570. Jensen ML, Auer RA (1988) Ketamine fails to protect against ischemic neuronal necrosis in the rat. Br / Anaesth
61:206-210.
Karkoutly C (1990) Die Prufung neuroprotektiver Arzneistoffwirkungen nach Vorderhirnischamie bei der Ratte und nach fokaler Ischamie bei der Maus. Dissertation, Marburg. Kemp IA, Foster AL, Wong EHF (1987) Noncompetitive antagonists of excitatory amino acid receptors. TlNS 10:294-298. Kirino T (1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239:57-69.
Krieglstein J (1989) Excitatory amino acids, adenosine and Neuronal calcium homeostasis-an overview. In Neurotransmission and Cerebrovascular Function II. Eds., J Seylaz, R Secombe. Elsevier Sci Pub B.V., pp. 441-453. Krieglstein J, Brungs H, Peruche B (1988) Cultured neurons for testing cerebroprotective drug effects in vitro. / Pharmacol Meth 20:39-46. Krieglstein J, Krieglstein C, Stock R (1972) Suitability of the isolated perfused rat brain for studying effects on cerebral metabolism. Naunyn-Schmiedeberg’s
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Krieglstein J, Sauer D, Nuglisch J, Karkoutly C, Beck T, Bielenberg GW, RoBberg C, Mennel HD (1989a) Calcium antagonists protect hippocampal neurons against ischemic damage. In: Alzheimer’s disease and related disorders. Eds., K Igbal, HM Wisniewski, S Winblad, A. Liss. New York, pp. 393-400. Krieglstein J, Sauer D, Nuglisch J, RoBberg C, Beck T, Bielenberg CW, Mennel HD (1989b) Naftidrofuryl protects neurons against ischemic damage. Eur Neural 29~224-228. Leander JD (1989) Tricyclic antidepressants block N-methyl-D-aspartic acid-induced lethality in mice. Br / Pharmaco/96:256-260. Lowry OH, Passonneau JV (1972) A Flexible System of Enzymatic Analysis. New York and London: Acad Press. Marietta MP, White PF, Pudwill CR, Way WL, Trevor AJ (1975) Self-induction of metabolism. / Pharmacol
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Nelson PC, Ruffner W, Nirenberg M (1969) Neuronal tumor cells with excitable membranes grown in vitro. Proc Nat/ Acad Sci USA 64:100& 1010.
Pauwels PJ, Opperdoes FR, Trouet A (1985) Effects of antimycine, glucose deprivation and serum on cultures of neurons, astrocytes and neuroblastoma cells. / Neurochem 44:143-148.
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B. Peruche and J. Krieglstein Peruche 6, Ahlemeyer B, Krieglstein J (1990) Cultured neurons for testing antihypoxic drug effects. / Pharmacol Meth 23:63-77. Pulsinelli WA, Brierley JB (1979) A new model of bilateral hemisphericischemiain the unanaesthetized rat. Stroke 10:267-272. Rischke R, Sauer D, Rogberg C, Mennel HD, Krieglstein J (1989) Vinpocetine ameliorates hippocampal neuronal damage following transient forebrain ischemia in the rat. In: Pharmacology of Cerebral lschemia 1988. Ed., J Krieglstein. StuttWiss Verlagsges, pp. 295-298. gart: Rod MR, Auer RN (1989) Preischemic and postischemic administration of dizocilpine (MK-801) reduces cerebral necrosis in the rat. Can/ Neural SC; 16:340-344. Roth-Schechter BF, Mandel P (1977) Preferential neurotoxicity of pentobarbital on nerve and glial cells in culture. fxperientia 33:627-630. Sachs L (1978) Angewandte lag, Berlin. Sauer D, Rischke HD, Bielenberg cetine prevents pocampus. Life
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R, Beck T, Rogberg C, Mennel GW, Krieglstein J (1988) Vinpoischemic cell damage in rat hipSci 43:1733-1739.
Schmidt J, Fischer HD, Wustmann C, Friedt A, Rudolph E (1989) Effectiveness and mode of action of nootropics and related drugs on posthypoxic transmitter release and learning behavior in rats. In: Pharmacology of Cerebral lschemia 1988. Ed., J Krieglstein. Stuttgart: Wiss Verlagsges, pp. 347-351. Seif el Nasr M, Sauer D, Rogberg C, Mennel HD, Krieglstein J (1989) Effects of NMDA-antagonists against neuronal damage after forebrain isch-
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Cells for Testing Neuroprotective
BARBARA PERUCHE AND JOSEF KRIEGLSTEIN
An attempt effects.
was made to use neuroblastoma
To achieve
blastoma containing Drugs
fresh
were
nutrient
medium.
of glucose.
available
damage,
than
naftidrofuryl,
were
ing with
under
induced cyanide
period
to recover
(1 mmol/L) the cyanide-
cells were
for another
to hypoxia
phosphates
drug
in neuro-
until
and culture
addi-
7 days.
24 hr after
protein
development
conof cell
and viability. and phenytoin 300 (*mol/L,
revealed protected
similar experimental drug
that neuroblastoma
neurotoxic
chlorpromazine,
and flunarizine
neuroprotective
suggested
this hypoxic
for the posthypoxic
damage. These results were comparable of neurons
of sodium
30 min prior
of high-energy
neuroprotective
was
after 6 hr by replacing
allowed
as representatives
barbiturates higher
During
They
hypoxia
by addition
to the cells from
determined cell activity
While
cells for testing
cytotoxic
and was terminated
Cell concentration
tent were
tine,
medium
deprived
hypoxia.
doses
damage,
cells after 10 days in culture
to the culture tionally
cellular
effects
effects
dizocilpine,
conditions. obtained
cells are suitable
from
In addition, from
applied
in
ketazo-
cells against hypoxic
neuroblastoma
to those obtained
when
ketamine, primary
cultures
they were
in keep-
in vivo experiments.
for testing
neuroprotective
It is drug
effects.
Key Words: Calcium
Cell
culture;
antagonists;
pressants;
Neuroblastoma
N-methyl-D-aspartate
cells;
Cytotoxic
(NMDA)
hypoxia;
antagonists;
Cyanide; Central
de-
Nootropics
INTRODUCTION Several in vivo models have been developed to investigate the consequences of ischemic and hypoxic damage of brain tissue (Krieglstein et al., 1972; Pulsinelli and Brierley, 1979; Tamura, et al., 1981; Kirino, 1982; Smith et al., 1984). To reduce the complexity of an in vivo model cultured neurons were used. Recently, an in vitro model has been developed in our laboratory for screening neuroprotective drug effects under hypoxic conditions by using neurons of chick embryo cerebral hemispheres as a primary cell culture system (Krieglstein et al., 1988). Cytotoxic hypoxia was induced by addition of sodium cyanide to the culture medium and several drugs revealed neuroprotection by preserving the neuronal ATP level, cell mass or viaFrom the lnstitut ftir Pharmakologie und Toxikologie, Philipps-Universitat Marburg, Ketzerbach 63, 3550 Marburg/Lahn, Germany. Address reprint request to: Professor J. Krieglstein, lnstitut fijr Pharmakologie und Toxikologie, Philipps-Universittit Marburg, Ketzerbach 63, 3550 Marburg/Lahn, Germany. Received December 3, 1990; revised and accepted March 15, 1991.
139 Journal of Pharmacological
Methods
8 1991 Elsevier Science Publishing
26, 139-148 W31) Co., Inc., 655 Avenue of the Americas, New York, NY 10010
140
6. Peruche and J. Krieglstein bility. The model
proved
of drugs (Krieglstein or not comparable For instance, their
character
(Breakfield,
for weeks,
the
protective
effects
with transformed
neuro-2a)
1976;
are no longer
the hypoxic of several
drugs
are experi-
they can be kept
However,
for limited
in order
they
of neurons,
needed.
conditions
cells arose.
to have preserved
et al., 1969);
cultures
using this cell
cell damage
to compare
effects
of whether
neuronal
are known
Nelson
than primary
and animals
line we had to redefine
the neuroprotective
et al., 1990). Now the question
cells (clone
much easier to handle
in culture
to demonstrate
results could be achieved
neuroblastoma
neuronal
mentally
to be suitable
et al., 1988; Peruche
them
and study
with
previous
results. MATERIALS
AND
METHODS
Materials All experiments
were
blastoma,
clone
American
Type Culture
and were
cultivated
carried
neuro-2a
out on cultured
(N2a,
Collection
in Falcon
tumor
passages 168-178). (ATCC,
culture
cells of type C-1300
The cells were
Flow Laboratories
Ltd., Irvine,
flasks (25 and 75 cm’;
neuro-
purchased
Serolab,
from
Scotland)
Aidenbach,
Germany). The following auxiliary materials were purchased from Gibco (Paisley, Scotland) : Dulbecco’s modified Eagle medium (DMEM) with sodium pyruvate and 1 g/L glucose,
Dulbecco’s
icillin/streptomycin tamine Puck’s
Eagle medium
(10000 units/ml
“special”
(without
and 10000 kg/mL,
glucose),
respectively),
penL-glu-
solution 200 mmol/L, nonessential amino acids, trypsine-EDTA solution in salt solution A (PBS). Fetal calf serum was supplied by Biochrom (Berlin,
Germany), albumin
modified
solution
sodium from
cyanide
from
Behringwerke
Merck
(Marburg,
(Darmstadt, Germany).
Germany) All other
and bovine
chemicals
serum
were
of re-
agent grade. chlorpromazine The following drugs were tested: Bayer, Leverkusen, Germany), dizocilpine maleate Harlow,
UK),
flunarizine
dihydrochloride
ketamine hydrochloride (Ketanesta, methanesulfonate (Sterling-Winthrop,
hydrochloride (MK-801,
(Sibelium@,
Parke-Davis,
Janssen,
Berlin,
Neu-lsenburg,
MSD,
(Megaphena, Terlings
Neuss,
Germany),
Germany),
Park
Germany), ketazocine
methohexital
so-
dium
(Brevimytal”, Lilly, Homburg, Germany), naftidrofuryl hydrogenoxalate (Duphenytoin sodium (Phenhydans, Desitin, Hamsodrile, Lipha, Essen, Germany), UCB Chemie, Kerpen, Germany), pyritinol burg, Germany), piracetam (NootropE, hydrochloride
(Encephabol@,
iurn@, Thiemann, Cultivation
Waltrop,
Conditions
Merck,
Darmstadt,
Germany),
vinpocetine
(Eusen-
Germany). of the Neuroblastoma
Cells
The cells were grown in 75 cm2 Falcon culture flasks. They were subcultured every 10 days, after reaching confluency. For hypoxic experiments, the cells were subcultured in 25 cm2 culture flasks. After trypsinization the cells were suspended in 20 mL culture medium and counted using a hemocytometer chamber. One mL (34 x IO5 cells) of this suspension was seeded into each 25 cm2 culture flask containing 5 mL of culture medium. As nutrient medium we used Dulbecco’s modified Eagle
Testing for Neuroprotective medium
with
2 mmol/L
sodium
glutamine,
pyruvate
and 1 g/L glucose
1% nonessential
amino
fortified
Drugs
by 10% fetal calf serum,
acids, and penicillin/streptomycin
(100
units/ml and 100 pg/mL, respectively). The cultures were kept in an incubator (Heraeus, Hanau, Germany) at 37°C in a humidified atmosphere of 95% air and 5% COZ. Cytotoxic Thirty replaced
Hypoxia
and Recovery
10 days of subculture
by adding incubation
a freshly prepared medium
6 hr the cyanide-containing
anide-free recover
Cells
min before hypoxia was started nutrient medium was aspirated off and by DMEM “special” (without glucose). Cytotoxic hypoxia was induced after
to the glucose-free After
of the Neuroblastoma
nutrient
medium
medium
(DMEM
stock solution
of sodium
was removed
and replaced
with 1 g/L glucose).
cyanide
of 1 mmol/L.
to give a final concentration
by fresh cy-
The cells were
allowed
to
for 7 days.
Application
of Drugs
Thirty min before glucose-free after hypoxia.
Control
Determination
the freshly
medium. cultures
Drugs
prepared were
drug solutions present
was stopped
Phosphates
and Protein
and metabolites
and
Passonneau
(1972)
using
the
until
volumes
to the 24 hr
of solvent.
Content
extracted
et al. (1988). ATP, ADP and AMP were measured
to Lowry
were added
in the medium
were treated with corresponding
of High-Energy
Cell metabolism Krieglstein
hypoxia
incubation
as already
described
enzymatically
Aminco-Bowman
by
according
fluorimeter
(Silver
Springs, MD). Energy charge (EC) was determined according to Atkinson (19681, i.e., EC = (ATP + 0.5 ADP)/(ATP + ADP + AMP). The protein content of the cultures
was determined
a protein
by the Biuret method.
Bovine serum albumin
was used as
standard.
Statistics Statistical
comparison
between
the drug-treated
sponding control group was performed by Duncan’s test (Sachs, 1978).
hypoxic
by one-way
groups
and the corre-
analysis of variance
followed
RESULTS Ten days after subcultivation was induced.
Transformed
a confluent
neuronal
cell sheet
had developed
cells are able to cover their
and hypoxia
energy
require-
ments under hypoxic/anoxic conditions by an increased anaerobic glycolysis provided that glucose supply is sufficient (Pauwels et al., 1985). Thus, to induce a deleterious energy failure in the cells as a basis for testing neuroprotective drug effects, it was necessary to combine hypoxia (incubation with NaCN) with glucose deprivation. Central depressant drugs like methohexital and phenytoin showed neurotoxic effects in doses higher than 300 kmol/L. They induced both a marked decrease in the ATP level and the energy charge of the neural cells as well as a loss of protein content of the cultures (Table 1).
141
142
B. Peruche and J. Krieglstein TABLE 1
Neuroprotective
Effect of Drugs Against Hypoxic Damage ATP
DRUG DRUG
CONC.
(kMOL/L)
NACN
CONC.
(MM~L~L)
(NMOL/MC
PROTEIN
ENERGY
PROTEIN)
(MC/FLASK)
CHARGE
Chlorpromazine
0 0.1 1 10 10 0
1 1 1 1 0 0
8.18 t 15.26 + 14.96 t 6.98 t 24.12 c 22.34 +
2.60 2.57** 3.22** 1.45 3.28 1.44
1.50 f 1.77 k 1.69 f 1.69 k 2.86 k 2.78 k
0.06 0.08** 0.19* O.fO* 0.78 0.29
0.87 0.85 0.85 0.87 0.97 0.96
r + + r c c
0.05 0.03 0.06 0.04 0.01 0.01
Dizocilpine
0 0.1 1 IO IO 0
1 1 1 1 0 0
13.19 2 15.29 * 16.09 2 16.55 k 27.82 f 27.50 2
1.60 1.10** l.OO** 0.48** 1.59 1.00
1.55 2 1.73 f 1.78 2 1.79 * 3.58 k 3.76 2
0.08 0.09* 0.13** 0.11** 0.42 0.07
0.91 0.92 0.93 0.94 0.98 0.97
c + r * ? 2
0.03 0.04 0.03 0.02 0.01 0.01
Flunarizine
0 0.1 1 10 IO 0
1 1 1 1 0 0
11.26 f 9.99 k 11.90 * 10.93 f 28.37 + 28.16 c
1.08 1.52 1.09 0.44 0.64 2.17
1.62 2 0.01 1.77 f 0.05' 1.85 IT 0.03"* 1.87 f 0.08** 2.81 k 0.28 2.79 c 0.17
0.85 0.84 0.85 0.85 0.97 0.95
+ 5 + k 2 +
0.01 0.03 0.05 0.02 0.02 0.01
1.55 1.42** 4.63" 0.93** 2.32 2.25
1.54 c 2.62 t 2.52 2 2.34 t 3.10 k 3.18 2
0.74 + 0.98 k 0.98 k 0.99 ? I?0 0.99 f
Ketamine
0 1 IO 100 100 0
1 1 1 1 0 0
10.11 20.05 21.04 20.00 30.79 30.26
Ketazocine
0 1 IO 100 100 0
1 1 1 1 0 0
16.15 ZL 1.37 20.09 k 1.52** 21.47 2 1.72** 23.05 f 1.89** 27.13 t 2.40 26.73 + 1.62
1.69 k 0.08 2.02 2 0.34** 2.04 2 0.28** 2.11 f 0.28** 3.10 IT 0.19 2.77 k 0.21
0.80 0.92 0.89 0.93 0.93 0.93
? 0.02 -c 0.01** " O.Ol** 2 0.03 2 0.02 k 0.02
0 100 300 1000 1000 0
1 1 1 1 0 0
17.01 + 0.86 16.74 2 0.94 16.70 i- 1.89 2.23 2 2.27** 30.68 2 1.10 28.65 k 2.58
1.94 f 0.14 1.79 If-0.07 1.71 + 0.10 0.32 + 0.36" 3.04 * 0.14 2.97 k 0.19
0.95 0.95 0.94 0.37 0.96 0.97
? i k i k k
1 1 1 1 0 0
18.54 k 20.25 k 18.33 2 19.80 2 27.01 k 27.23 +
0.89 1.23 1.15 1.64 1.59 1.34
1.19 I! 0.06 1.48 k O.lO** 1.54 k 0.11** 1.49 2 0.09** 2.77 2 0.26 2.87 k 0.24
0.88 0.92 0.93 0.92 0.94 0.94
k 0.02 k 0.0-l k 0.03 2 0.02 ? 0.02 -c 0.01
1 1 1 1 0 0
12.85 + 12.53 k 12.71 k 8.71 2 20.90 k 27.10 2
2.34 1.20 2.02 1.71** 1.73 2.10
1.53 2 0.09 1.59 ?I 0.06 1.53 k 0.10 0.75 " 0.15** 2.59 * 0.04 2.64 f 0.04
0.86 0.89 0.84 0.58 0.92 0.95
* 0.04 -c 0.05 * 0.05 k 0.04** k 0.02 -c 0.01
Methohexital
Naftidrofuryl
Phenytoin
0 0.1 1 IO IO 0 0 IO 100 1000 1000 0
k 2 k 2 k 2
0.17 0.19** 0.16** 0.38** 0.12 0.16
0.05 0.01" O.Ol** 0.04** 0.01
0.01 0.01 0.03 0.41" 0.01 0.0-I
Testing for Neuroprotective TABLE 1
Drugs
(continued) ATP DRUG CONC.
DRUG
NACN
(,.LMOL/L)
2 1.37
1.40
*
0.29
0.88
?
0.01
13.12
k 1.56
1.66
*
0.15
0.87
?
0.03
1000
14.14
2 2.62
1.44
2
0.31
0.82
"
0.11
10000
13.93
? 2.39
1.70
"
0.18
0.85
2
0.09
0
26.97
t
1.90
2.74
2
0.12
0.94
-+ 0.01
0
27.41
2 2.12
2.58
?
0.21
0.90
2
0.04
10.58
t
0.45
1.61
2
0.05
0.73
*
0.02
10.31
5 0.42
1.54
+
0.08
0.73
2
0.02
100
11.05
k 0.73
1.57
+
0.08
0.71
?
0.06
1000
12.53
t
1.69
1.55
?
0.08
0.73
"
0.06
0 0
1
IO
1000
0
29.44
2 1.40
2.92
+
0.20
0.90
?z 0.01
0
0
29.70
t
2.01
2.79
L
0.11
0.92
2
0.02
0
1
16.22
5 2.17
1.72
?
0.40
0.94
*
0.03
13.55
? 1.96
1.88
?
0.26
0.93
*
0.02
10
12.02
2 1.61
1.83
?
0.34
0.91
*
0.03
100
13.69
-+ 3.11
1.47
*
0.43
0.89
"
0.05
Vinpocetine
Neuroblastoma
100
0
30.88
2 0.63
2.87
?
0.26
0.95
2
0.01
0
0
27.86
k 3.85
2.88
?
0.15
0.97
2
0.01
ceils were
incubated
Recovery
period
for 6 hr with NaCN in glucose-containing
to the cells from 30 min prior
experiments. way analysis
For the statistical of variance
in all applied
hypoxic
as was the protein tures. This could
(NMDA)
content
revealed
and untreated
hypoxic
dizocilpine,
compared
agonist. cells with
as indicated
were
5 SD of six cultures
ketamine
one-
and chlor-
neuroprotective
with
hypoxic
of hypoxic
nootropics
their posthypoxic
after 10
Drugs
** P < 0.01.
significant
after incubation
naftidrofuryl induced a significant increase calcium antagonist flunarizine significantly cell cultures
medium
lasted 7 days.
are given as means
* P < 0.05;
antagonists
of the cultures
also be observed
did not ameliorate
of the neural
Values
drug-treated
applied:
in glucose-free
medium
The ATP level of the cells was significantly
with ketazocine, a kappa-opioid Treatment of neuroblastoma vinpocetine
between
test were
concentrations
cell damage.
(1 mmol/L) nutrient
until 24 hr after hypoxia.
comparison
and Duncan’s
The N-methyl-D-aspartate promazine
ENERGY CHARGE
14.28
Pyritinol
days of cultivation.
PROTEIN (MC/FLASK)
0
10000
against
PROTEIN)
(MM~L/L)
100
Piracetam
available
(NM~L/M~
CONC
nontreated
neuroblastoma
like piracetam,
recovery,
effects elevated,
whereas
pyritinol incubation
culcells and with
in protein content of the cultures. The ameliorated posthypoxic development
by an increased
protein
content.
DISCUSSION In the present study we tried to establish a model of a hypoxic neural cell line for testing neuroprotective drug effects comparable to the above mentioned model of primary neural cell cultures deriving from chick embryo cerebral hemispheres and Krieglstein, 1989; Peruche et al., 1990). (Krieglstein et al., 1988; Ahlemeyer Neuroblastoma
cells (clone
neuro-2a)
were
isolated
from a mouse
tumor
and have
143
144
B. Peruche and J. Krieglstein been cultured
in vitro since 1967 (Augusti-Tocco
primary cell culture system, manageable in vitro model. Despite
and Sato, 1969). In contrast
the cell line in general
their transformation
represents
these cells are able to preserve
a vigorous
to the
and more
their neural character
(Nelson et al., 1969; Breakfield, 1976; Willow et al., 1983), an indispensable requirement for the comparison of the results obtained in the present study with those obtained cultures
under
(Krieglstein
similar
experimental
conditions
et al.., 1988; Ahlemeyer
by using primary
and Krieglstein,
1989;
neural
Peruche
cell
et al.,
1990). The question
arose as to whether
against hypoxia and whether in a similar known
manner
e.g.,
glycolysis.
(Pauwels
we defined
Incubation
of the cells with
recovery in nutrient about 50% of control (120 min)
recovery
complete
cell death
The influence the neural
a marked
or neurons
Pasteur-effect
requirements
as an adaptation experimental
NaCN
(1 mmol/L)
a rapid breakdown
sensitivity
dep-
anaerobic
as long as glucose with glucose
for neuroblastoma
in glucose-free
of energy
It is well
under oxygen
in combination conditions
by drugs
in vivo.
by an increased
to hypoxia
et al., 1985). Using NaCN
ischemia-like
of 6 hr induced
cells reflect a similar against hypoxic damage
cell cultures
cells exhibit
This can be interpreted
deprivation duration
neural
the cells cover their energy
supply is sufficient cells.
as primary
that neuroblastoma
rivation,
neuroblastoma
they can be protected
metabolism.
medium
for a
After 7 days of
medium a restoration of cellular activity and cell growth to was measurable (Figure I). Following shorter hypoxic periods
was accomplished
within
2 days while
12 hr of hypoxia
caused
(data not shown).
of various
drugs on the posthypoxic
cells was determined
by measurement
Posthypoxic
permd
recovery
and development
of the high-energy
of
phosphate
Iweeks)
FIGURE 1. Development of neuronal damage after 6 hr of cytotoxic hypoxia and a recovery period of 2 weeks. After 10 days in culture neuroblastoma cells were incubated with 1 mmol NaCN/L in glucose-free medium for 6 hr. Development of hypoxia-induced neuronal damage was determined during a posthypoxic recovery period of 2 wk. Protein content of the cultures was measured as a reliable indicator of cell growth and viability according to the Biuret method. Data are given as means +SD of six experiments. For the statistical comparison different from the correone-way analysis of variance and Duncan’s test were applied: “P < 0.01, different from the protein content measured imsponding normoxic control: mediately after hypoxia: hP < 0.01.
Testing for Neuroprotective concentration
of the cells and protein
content
of the cultures.
Drugs
Drugs were available
to the cells from 30 min before until 24 hr after hypoxia. It is assumed that oxygen deficiency induces a massive influx of calcium as well as an increased release of excitatory
amino acids both of which
of pathologically Wieloch,
disturbed
1985; Goldberg
induce
metabolic
neural cell death at the end of a cascade
reactions
in vitro and in vivo (e.g.,
et al., 1987). Therefore
we tested
Siesjo and
drugs that are believed
to interrupt these deleterious events, e.g., centrally depressant drugs, NMDA antagonists and calcium antagonists, as well as substances belonging to the group of the so-called
nootropics.
Barbiturates in therapeutic dosages are known to lower energy the cells and thus protect them against hypoxia-induced depletion and subsequent effects
cell death.
in doses higher
by Ahlemeyer
and Krieglstein
experimental
hypoxic
strated chronic
In the present
than 300 pmol/L,
study methohexital
which
is in keeping
(1989) using primary
conditions.
revealed with
neural cell cultures
Roth-Schechter
and Mandel
neurotoxic
results described under
similar
(1977) also demon-
selective neurotoxic effects of pentobarbital (IO pmol/L-I mmol/L) after incubation of mixed cultures of neurons and glia. The differences in the
sensitivity
of these cells to pentobarbital
metabolism. Long-term uncoupling
Neural
cells are more
incubation
with
of oxidative
are explained
sensitive
pentobarbital
in doses
phosphorylation
by differences
to oxygen
et al. (1983).
These
deleterious
effects
deficiency
higher
and thereby
in their energy than glial cells.
than 10 Fmol/L
causes cell damage.
rotoxicity of phenytoin, its influence on ion homeostasis and calcium permeability of the neural cell membrane Swaiman
requirements of of energy stores
induces The neu-
and especially on sodium have been described by
of the drug could
be responsible
for the posthypoxic neural cell damage observed in the present study. Dizocilpine, ketamine and chlorpromazine were able to inhibit hypoxia-induced depletion
TABLE 2
of energy
stores of the cells and protein
loss of the cultures
in all applied
Comparison of Drug Effects In Vitro and In Vivo
Chlorpromazine Dizocilpine Flunarizine Ketamine Ketazocine Methohexital Naftidrofuryl Phenytoin Piracetam Pyritinol Vinpocetine
+ + + + + _
+
+
+ + + + -
+ + -
+ -
+ _ -
+ -
+ -
+ -
+ + +
(Leander, 1989) (Seif el Nasr et al., 1989) (Krieglstein et al., 1989a) (Seif el Nasr et al., 1989) (Tang, 1985) (Karkoutly, 1990) (Krieglstein et al., 198913) (Karkoutly, 1990) (Schmidt et al., 1989) (Schmidt et al., 1989) (Sauer et al., 1988)
+ = neuroprotective effect; - = no effect demonstrable * the results obtained with primary neuronal cell cultures are described by Ahlemeyer and Krieglstein (1989)
145
146
B. Peruche and J. Krieglstein concentrations, tures
which
(Ahlemeyer
promazine
coincides
with results obtained
and Krieglstein,
revealed
1989).
neuroprotective
brain ischemia
of the rat by reducing
hippocampus
formation
of action
the blockade
discussed
(Kemp
itive NMDA
action in histological
(Krieglstein,
1989;
of the calcium
antagonists
binding
receptor
from primary
ketamine,
neural cell damage operated
cell cul-
and chlor-
studies in vivo after fore-
in the selectively 1989).
vulnerable
As a mechanism
by the NMDA
and dizocilpine
are known
to the PCP recognition
site, which
and to the NMDA-gated
neural
dizocilpine
Rod and Auer,
channel
et al., 1987). Ketamine
linked to the NMDA
Except
Ca*+ channel.
receptor
is
as noncompetis allosterically Ketamine
failed
to protect neurons against ischemic damage in vivo (Jensen and Auer, 1988) probably because of its short plasma half life (IO min in rats) (Marietta et al., 1975). Chlorpromazine
is also believed
to interact
with the NMDA-receptor-complex
binding site (Leander, 1989). The in vitro application of ketazocine, protective
effects
in the neural
after hypoxia
(Ahlemeyer
demonstrated
in other
studies.
lwama
a kappa-opioid
cell line as well
and Krieglstein, in vitro
agonist,
as in primary
at the Zn*+ induced
neural
1989). This neuroprotection
(Iwama
et al., 1987) as well
et al. (1987) suggest this substance
neuro-
cell cultures has also been
as in vivo (Tang,
influences
a subtype
1985)
of the so-
dium channel and thus an inhibition of neural activity. As for neuroblastoma electrical membrane activity has been shown (Willow et al., 19831, this could possible
explanation
for the amelioration
the present study. The calcium antagonist effect
also observed
system. This compound by interaction
with
flunarizine
by Ahlemeyer
inhibited to inhibit
binding
prevent consecutive cell death. Nootropics like pyritinol, piracetam tective
effects
in the present
state of the cells obtained
protein
and Krieglstein
is supposed
intracellular
of energy
loss of the cultures
(1989) in a primary
the increase
in cytosolic
sites (van Zwieten,
and vinpocetine
study. This coincides
cells be a in
used, an
cell culture Ca*+
levels
1986) and thus
did not reveal
may
neuropro-
with the results of the long-term
study with primary cultures of neurons described by Ahlemeyer and Krieglstein (1989), except pyritinol, which inhibited protein loss in the primary neural cell culture system. The disagreement not be explained
because
between
results obtained
the mechanism
with pyritinol
of the neuroprotective
in vitro can-
action
for this
drug is unknown. While the vincamine derivative vinpocetine could reduce the development of neural damage after forebrain ischemia in the rat (Sauer et al., 1988), the substance
failed
vitro systems,
probably
to reveal because
neuroprotective of an indirect
effects
in the abovementioned
adenosine
agonistic
effect
in
(Rischke
et al., 1989). Naftidrofuryl is known to inhibit neural damage in vitro (Ahlemeyer and Krieglstein, 1989) and in vivo (Krieglstein et al., 1989b). Protein loss of the neuroblastoma cell culture used in the present study was also inhibited by naftidrofuryl. For this drug action (Fujikara In conclusion,
antiserotonergic et al., 1989). neuroblastoma
effects
are suggested
cells were
as a possible
demonstrated
mechanism
to be suitable
of
for testing
neuroprotective drug effects. The results obtained with this cell line were comparable to those obtained from primary cultures of neurons and even from in vivo
Testing for Neuroprotective
Drugs
models of cerebral ischemia (Table 2). Moreover, neuroblastoma cells seem to be advantageous because they are vigorous and more manageable than primary cultures of neurons. This work was supported by a grant from the Bundesminister fur Forschung und Technologie (Bonn). The authors are indebted to Ms. R. Seidel and Ms. S. Engel for their skillful technical assistance.
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