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Journal of Cerebral Blood Flow and Metabolism
12:873-876 © 1992 The International Society of Cerebral Blood Flow and Metabolism
Published by Raven Press, Ltd., New York
Short Communication
Intracerebral Microdialysis of Extracellular Amino Acids
in the Human Epileptic Focus
*Elisabeth Ronne-Engstrom, *tLars Hillered, tRoland Flink, *Bo Spannare,
§Urban Ungerstedt, and *Hans Carlson
*Department of Neurosurgery, tDepartment of Clinical Chemistry, and tDepartment of Clinical Neurophysiology,
University Hospital of Uppsala, Uppsala; §Department of Pharmacology, Karolinska Institute, Stockholm, Sweden
creases of the extracellular ASP, GLU, GLY, and SER
concentrations were observed. The other amino acids an­
alyzed, including TAU, showed small changes. The re­
sults support the hypothesis that ASP, GLU, GLY, and
possibly SER, play an important role in the mechanism of
seizure activity and seizure-related brain damage in the
human epileptic focus. Key Words: Brain-Human­
Epilepsy-Microdialysis-Amino acids-Excitotoxicity.
Summary: Extracellular levels of aspartate (ASP), gluta­
mate (GLU), serine (SER), asparagine (ASN), glycine
(GLY), threonine (THR), arginine (ARG), alanine (ALA),
taurine (TAU), tyrosine (TYR), phenylalanine (PHE),
isoleucine (ILEU), and leucine (LEU) were monitored by
using intracerebral microdialysis in seven patients with
medically intractable epilepsy, undergoing epilepsy sur­
gery. In association with focal seizures, dramatic in-
Epileptic discharges may result from a mismatch
PATIENTS AND METHODS
between excitatory (e.g., aspartate and glutamate)
Patients
and inhibitory (e.g., -y-aminobutyrate, GABA)
Seven patients with medically intractable epilepsy,
aged between 10 and 48 years were investigated. Six had
complex partial seizures and one patient simple partial
seizures. The preoperative evaluations included 24 h/day
video-EEG monitoring using subdural electrodes (Blom
et ai., 1989), positron emission tomography (PET) with
fluorodeoxyglucose, magnetic resonance imaging (MRI),
computer-assisted tomography (CAT) and neuropsycho­
logical investigations. Anterior medial temporal lobe re­
section including the hippocampus was performed in
three patients. Partial cortical resection of the frontal lobe
was performed in another three patients and one patient
was subjected to a midtemporal cortical resection sparing
the hippocampus. The patients were under general anes­
thesia using propofol, fluothan, fentanyl, succinylcholine,
oxygen, and nitrous oxide. No changes of the individual
antiepileptic pharmacotherapy were made prior to the
surgical procedure. Cortical dysplasia was found in one
patient, gliosis in the hippocampus or in the cortex in five
patients, and a gliomatous tumor in the patient with sim­
ple partial seizures.
amino acid systems (Brazier, 1974; Meldrum, 1984).
Excitatory amino acids could also act as causal fac­
tors in seizure-related brain damage, according to
the concept of excitotoxicity (Olney, 1985; Choi,
1988). In order to investigate biochemical and elec­
trophysiological relationships in the human epilep­
tic focus, microdialysis (Delgado et aI., 1972; Un­
gerstedt and Pycock, 1974; Ungerstedt, 1984) was
used in the present study together with electrocor­
ticography (ECoG).
Received December 10, 1991; final revision received March
18, 1992; accepted March 18, 1992.
Address correspondence and reprint requests to Dr. Elisabeth
Ronne-Engstrom at Department of Neurosurgery, University
Hospital, S-75 1 85 Uppsala, Sweden.
Abbreviations used: CAT, computer-assisted tomography;
ECF, extracellular fluid; ECoG, electrocorticography; GABA,
,,(-aminobutyric acid; MRI, magnetic resonance imaging; PET,
positron emission tomography.
This work was presented in part at the XVth International
Symposium on Cerebral Blood Flow and Metabolism, Miami,
Florida, June 7-9, 1991.
Preoperative neurophysiological procedures
In order to verify the exact location of the epileptic
focus, a standard clinical protocol was employed involv­
ing recording and electrical cortical stimulation by con­
ventional electrophysiological techniques. The ECoG
873
E. RONNE-ENGSTROM ET AL.
874
was recorded during the entire procedure and evaluated
for interictal epileptiform activity, spontaneous seizure
activity, electrically induced afterdischarges or seizure
activity. Epileptiform rhythmic spike-wave activity elic­
ited by electrical stimulation was considered as afterdis­
charges when confined to the electrode closest to the
stimulation point. If the afterdischarges spread to adja­
cent electrodes and were followed by sudden slowing in
the ECoG, this was instead classified as an electrically
induced seizure with a postictal suppression.
during the equilibration period and reached basal
levels within the first 30 min. The basal levels did
not differ between the frontal and temporal regions.
Levels of amino acids in relation to seizures and
cortical afterdischarges
Seizures. Three patients developed altogether
five episodes with seizure patterns after electrical
stimulation and another patient exhibited five epi­
sodes with spontaneous seizures. No generalization
Microdialysis
During the neurophysiological procedure sampling
from the extracellular fluid (ECF) was performed with
sterilized CMA/lO probes (CMA Microdialysis, Sweden)
with membrane lengths of 6 or 10 mm. The probe was
inserted into the epileptogenic zone, as defined by ECoG,
and perfused with sterile Ringer solution at a flow rate of
5 ILUmin. An equilibration period of 30-40 min was al­
lowed to obtain basal amino acid levels (Hillered et aI.,
1990). Sampling was then performed in 2-min fractions.
The dead space in the probe and the outlet tubing was
7-10 ILL The results presented are corrected for dead
space. The samples were analyzed for amino acids by
HPLC according to Lindroth and Mopper (1979) as de­
scribed by Tossman and Ungerstedt (1986). The HPLC
system was optimized for analyses of aspartate and glu­
tamate at the expense of GABA and glutamine. Lactate
and pyruvate were measured by HPLC with UV detec­
tion (Hallstrom et aI., 1989).
Ethics
The study was approved by the ethics committee at
University Hospital of Uppsala, Sweden. The patients
gave their informed consent to the measurements.
of the seizures was recorded.
Sampling from the ECF could be obtained in
eight of the ten seizure periods. Altered levels of
amino acids were detected in relation to the spon­
taneous as well as the electrically induced seizures.
Aspartate displayed the largest increases ranging
from 1. 3 to 79. 0 times the basal levels. Glutamate
increased between 1. 8 and 16. 2, serine between 1.5
and 8. 8, and glycine between 1.4 and 2 1. 1 times the
basal levels (Fig. O. The elevations were observed
within 2 min from the onset of the seizure pattern in
the ECoG and returned to basal levels within 6 min.
The other amino acids displayed minor changes,
i.e., less than two times the basal level.
Lactate and pyruvate were measured in four pa­
tients, during two seizures. Lactate increased 1. 5-
1. 7 times the basal level and the lactate/pyruvate
ratio increased from a basal level of 19 to 24-26.
Afterdischarges. In 29 of a total of 45 episodes of
afterdischarges, biochemicql monitoring was per­
formed. Minor or no changes of amino acid concen­
RESULTS
trations were detected (Fig. O. Lactate and pyru­
vate were analyzed in three patients with afterdis­
Basal levels of amino acids
Table 1 gives the basal levels of amino acids in
the dialysates from all the patients. Since the only
charges and the relative changes ranged from 0.8-
1. 2 times the basal levels.
difference between the probes used in patient 1 and
2 and in the rest of the cases was the membrane
DISCUSSION
length (6 vs. 10 mm), the results from the first two
patients were corrected by a factor of 0. 7 (Lindefors
In this series of measurements dramatic eleva­
et aI, 1989) to allow a direct comparison. High
tions of aspartate and glutamate accompanied both
amino acid levels were found immediately after in­
spontaneous and electrically induced seizures. This
sertion of the probe. The concentrations decreased
is the first demonstration of such pronounced in-
TABLE 1. Basal levels (ILM) of amino acids in the epileptic focus
Patient
ASP
GLU
ASN
SER
1
2
3
4
5
6
7
Mean
SD
0.04
0. 15
0.24
0.08
0.23
0. 10
0.16
0. 14
0.07
0. 10
0.35
0.69
0. 10
0.72
0.26
0.74
0.42
0.29
0.04
0.16
0.23
0. 17
0.39
1. 18
0.15
0.33
0.39
0.32
1.33
1.47
0.87
2. 12
6.70
1.04
1.98
2.15
GLY
TRR
ARG
ALA
TAU
TYR
P RE
ILEU
LEU
2.96
1.0 1
0.64
0.94
1.23
0.37
1. 19
0.92
0. 18
0.3 1
0.39
0.24
0.65
3.66
0.6 1
0.86
1.25
0. 18
0.70
0.35
0.85
0.72
1.0 1
0.29
0.59
0.3 1
0.42
1.06
0.83
0.68
1.32
18.36
0.42
0.33
6.65
0. 19
1.04
1.38
0.78
1.77
4.75
0.88
1.54
1.50
0. 1 1
0.54
0. 16
0.18
0.42
0.90
0.20
0.36
0.29
0.13
0.45
0.30
0.22
0.47
1. 13
0.20
0.4 1
0.34
0.08
0.4 1
0.14
0. 12
0.24
0.92
0. 10
0.29
0.30
0. 13
0.68
0.32
0.32
0.6 1
1.8 1
0.24
0.59
0.57
The basal levels were calculated as the mean value of the concentrations in three consecutive 5-min fractions at the end of the
equilibration period.
J Cereb Blood Flow Metab, Vol. 12, No.5. 1992
MICRODIALYSIS IN THE HUMAN EPILEPTIC FOCUS
•
875
SEIZURES
(79.18)
Gi
>
Q)
- 25
iii
UI
ttl
.Q 20
�
�
.:.:
FIG. 1. The ratio of peak concentrations
to the basal level for amino acids in the
human epileptic focus during seizures (n
8) and afterdischarges (n
29). Col­
umns represent average values and dots
individual values. The stimulation param­
eters were 50 Hz biphasic square wave
pulse, with a pulse duration of 1-2 ms, an
interphase interval of 1 ms, pulse train du­
ration of 2-4 s, and a current intensity of
5-15 mAo The duration of spontaneous
seizures varied between 40 and 125 sec,
electrically induced seizures between 18
and 80 sec, and the duration of afterdis­
charges between 4 and 28 S.
=
�
15
10
Co
=
ASP
Q)
>
GLY GLU SER ASN THR ARG ALA TAU TYR PHE ILEU LEU
5
AFTERDISCHARGES
�
�
ttl
.Q
4
Gi 3
>
Q)
�I 6nnOODCJoOooon
ASP
GLY GLU SER ASN THR ARG ALA TAU TYR PHE ILEU LEU
creases in extracellular excitatory amino acids in
1992). The results argue against a severe energy dis­
association with seizure activity in the human epi­
turbance being the mechanism of amino acid re­
leptic focus (cf. During et aI., 1989 and Do et aI.,
lease, at least in focal seizures with a short dura­
1990). Marked elevations of extracellular serine and
tion.
glycine were also found. Serine is not presently re­
garded as a neuroactive amino acid, but it is inter­
Methodological considerations
convertible with glycine in the central nervous sys­
During seizure activity, a substantial decrease
tem (McGeer and McGeer, 1989). Apart from being
an inhibitory neurotransmitter, particularly in the
(20-50%) of the extracellular space may occur
(Heinemann, 1986). A rapid decrease of the extra­
spinal cord, glycine may play an important role
cellular volume may cause transiently increased
by potentiating glutamate-mediated brain injury
concentrations of substances remaining in this com­
(Uckele et aI., 1989) via the glycine modulatory site
partment. On the other hand, the diffusion of a
on the N-methyl-D-aspartate receptor (Choi, 1988).
given substance in the brain may change by a de­
Therefore serine and glycine may play a role in the
crease of the ECF fraction, considerably lowering
pathophysiology of human epilepsy.
mass transport into the dialysate (Benveniste, 1989;
Regarding the time course of the changes of the
Lindefors et aI., 1989). Until the net effect of these
amino acid levels, our results showed that there was
phenomena are better understood we regard any
an increase within minutes, in association with the
increase in the dialysate concentrations of amino
seizures. The decrease and return to preseizure lev­
acids below a factor of two as potentially insignifi­
els was also within the same range of time.
cant in terms of altered release/uptake.
Lactate and pyruvate were analyzed in two sei­
Drainage of substances (e.g., glucose) is a poten­
zures. The changes observed were small compared
tial problem with the microdialysis technique, as
with those in patients with cerebral ischemia or
suggested by results from measurements in adipose
trauma (Hillered et aI., 1990; Persson and Hillered,
tissue (Jansson et aI., 1990). The possible effects of
J Cereb Blood Flow Metab, Vol. 12, No.5, 1992
E. RONNE-ENGSTROM ET AL.
876
glucose drainage in the brain are to a large extent
unexplored. However, since previous measure­
ments using intracerebral microdialysis have shown
marked increases (4-8-fold) of lactate in response to
ischemia (Hillered et al., 1990) a depletion of glu­
cose does not seem to occur in the human brain in
acute measurements.
The present investigation using the combination
of electrophysiological methods and intracerebral
microdialysis, is the first to provide direct evidence
of dramatic elevations of extracellular aspartate,
glutamate, serine, and glycine in association with
both spontaneous and electrically induced seizures
in man. The results support the hypothesis that as­
partate, glutamate, glycine, and possibly serine,
play an important role in the mechanism of seizure
activity and seizure-related brain damage in the hu­
man epileptic focus.
Acknowledgment: The authors are grateful to Ms. Bodil
Kaller for skillful technical help. The study was sup­
ported by the Margarethahemmet Foundation, the Ulf
Lundahls Foundation, and the Swedish Medical Research
Council.
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