60
Microdialysis Study
Abstrak
Tujuan penelitian ini adalah untuk mengetahui perubahan metabolisme neuro
transmitter dan bahan-bahan metabolitnya setelah pemberian ATX-III ke dalam
striatum melalui penggunaan teknik mikrodialisis.
Tikus jantan dewasa merupakan subjek penelitian microdialisis secara in vivo
pada jaringan otak. Stariatum otak dilakukan kanulisasi pada binatang percobaan
yang telah dianastesi. Setalh 20-24 jam pada binatang percobaan diberikan cairan
Ringer (2 ul/menit) pada keadaan istirahat. Larutan ini pada awalnya tanpa
mengandung ATX-III dan kemudian dilanjutkan dengan ATX-III (10 ug/ml).
Larutan diberikan minimal 50 menit untuk mempertahankan kondisi stabil pada
hewan coba. Micro dialisis otak dan HPLC (ECD-100, EICOM Co., Kyoto)
dilakukan dengan sistim otomatis. Kolum HPLC adalah MA-50DS (4.6 x 150
mm), fase mobil adalah 0.1 M buffer sodium sitrat-asetat (pH 3.5) yang
mengandung 0.1 ml EDTA dan 17% methanol. Larutan ini dikumpulkan dengan
interval 25 menit dan dimasukkan secara langsung ke dalam HPLC-ECD.
Data yang khas akan didapatkan setelah 50 menit perlakuan. Kandungan
metabolit Dopamin (DA) seperti DOPAC DOPAC (3,4-dihydroxyphenyl acetic
acid), HVA (homovanillic acid) and HMPG (4-hydroxy-3-methoxy
phenylethylene glycol) akan menurun setelah injeksi ATX-III ke dalam stritum
otak. Metabolit Serotonin (5HT=5-hudroxytriptamine, 5 HIAA (5hydrxyindoleacetic acid), juga akan menurun setelah pemberian ATX-III. Tetapi
kadar serotonin, DA dan NE (Nor epinefrin) akan meningkat setelah pemberian
ATX-III.
Kadar serotonin yang tinggi, DA dan NE setelah pemberian ATX-III secara
langsung ke dalam ventrikel otak memainkan peranan penting terhadap terjadinya
perdarahan paru pada hewan coba.
kata kunci ; asebotoxin, brain, microdialysis
INTRODUCTION
Ericaceous family plant produced
some toxic substances, a di terpenoids,
with
perhydroazulene
skeleton.
Asebotoxin-III was found as a
diterpenoid isolated from the leaves of
asebi, Pieris japonica.(1)
Death of
enimal experiment from this toxin could
occur by various ways such as: cardiac
arrest,
respiratory
failure,
hypersecretion,
hypotension
and
convulsion.
Studies
of
ATX-III
which
introduced into cerebral ventricles of
guinea pig produce hemorrhage into
alveolar cavities of the lung.(2) The
proposed
that
possibly
massive
symphatetic discharge will stimulate
hypothalamus. However this hypothesis
need to be tested to clarify the exact
mechanism on the alteration of brain
metabolism of autonomic nervous
system.
The Harada N(3) concluded that on
the mechanisms for the lung hemorrhage
induced by ATX-III because of
depolarization of some neurons in the
hypothalamus guinea pig. He also
proposed that the massive symphatetic
Majalah Kedokteran Andalas No. 2. Vol.23. Juli- Desember 1999
61
Microdialysis Study
discharge will produce severe pulmonary
hypertension and hemorrhage.
Other researfhers, Maekawa et al.
fund that ATX-III induced pulmonary
hemorrhagic edema colud be prevented
by inhibiting the depolarization of nerve
cell membrane of symphatetic centre in
the hypothalamus of the brain rat.(4)
Studies on the action of ATX-III on
sodium channel were reported that ATXIII will produce a marked depolarization
on the squid nerve do to a specific
increased
in
resting
sodium
permeability.(5) Later, Seyama and
Narahshi (6,14) found that grayanotoxin
molecule caued a slow opening the
sodium channel of squid nerve
membranes. It was known that ATX-III
was the most cardiotripic and toxic
compound among the member of
grayanoid toxin (7) suggested the
grayanotoxin opened Na channels from
inside the squid axonal membrane.
Takeya et al. also concluded thad
GTX-I (grayanotoxin) as a Ericaceous
family toxin increases the resting Napermeability on the papillary muscles
isolated from the guinea pig heart.
Recently, Hotta et al. found that ATX-III
increases net Na(+)-influx via Na+
Channel in guinea pig erythrocytes and
heart respectively.(2,11-13)
Furthemore, Maekawa et al.
reported
that
ATX-III-induced
pulmonary hemorrhage edema could be
prevented
by
inhibiting
the
depolarization of nerve cell membrane of
symphatetic center in the hypothalamus
of the brain rat.(4)
The alterations on brain metabolism
could be followed by microdialysis
method for measuring the extracellular
fluid of neurotransmitters and its
metabolites in animal experiment.
Neurotransmitters and its metabolites
were collected into the striatum or other
brain regions. Nakano and Mizuno (in
press 1995) suggested that striatal
dopaminergic neuronal function reaches
a maximum level at 2-3 months old
rats.(15-22)
Dopamine (DA) is a brain
neurotransmitter and precursor in the
biosyinthesis of nor epinephrine (NE|) in
post ganglionic Symphatetic fibres.
Synthesis of NE within the terminals of
adrenergic axons is associated with
precursor DA in the axons. DA is
metabolized with monoamine oxidase
(MAO)
and
catechol-o-methyl
transferase (COMT). DOPAC (3,4dihydroxyphenylacetic acid) was derived
from a newly synthesized pool or DA.
Serotonim (5-hydroxytryptamine = 5
HT) was a serum vasoconstrictor
substance. Serotonin-, NE- and DAcontaining neurons have been shown to
project to the mammalian spinal cord.(25)
Recently, Kobayashi and Amenta
reported that neurotransmitter receptors
in the pulmonary endothelium heve an
importance
role
in
the
pathopyysiologicel changers in the
pulmonary circulation.(23)
The aims of this study was to
investigate
the
alterations
of
neurotransmitter receptors metabolism
and its metabolites after administration
of ATX-III into the brain ventricle by
using microdialysis technique.
MATERIALS AND METHODS
Animals:
Adult male Sprague-dawley rats were
obtained fram Japan SLC, Inc.
(Hamamatsu, Japan). The animal were
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62
Microdialysis Study
kept standard food and water adlibitum
under a 12 hours light-dark cycle (lights
on at 7:00 a.m). Experiments were
started between 9:00-11:00 a.m. The
animal experiments were conducted
according to the guide-lines for animal
experiment prescribed by the Aichi
Medical University.
Microdialysis:
Rats were anesthesized with sodium
pentobarbital (Nembutal;50mg/kg body
weidht and placed in a stereotactic
apparatus. The skull was exposed, and
small hole was drilled to allow
implantation of a guide cannula into the
striatum and nucleus accumbence.
Stereotactic coordinates from bregma
were A+1.0, L+2.5, V-4.0 (dura) for
striatum and A+2.5, L+1.5, V-6.0 for
nucleus accumbence consequtively
according to the skull screw and dental
cement. Microdialysis probe (BDP-I-803; molecular cut-off 50,000;- EICOM
Co., Kyoato, Japan) were designed to
extend 3 mm beyond the tip of the guide
cannula.
HPLC analysis:
Brain microdialysis and HPLC
(High
Performance
Liquid
Chromatography) were perfomed with a
fully automated system. Extracellular
contents of neurotransmitters and its
metabolites were detected with an
electrochemical detector (ECD-100,
EICOM Co., Kyoto). The dialysis probe
was perfused with Ringer's solution
(NaCL 148 mM, KCL 4.0 mM, CaC12
2.3 mM) at a flow rate of 2 ul/min by
means of a high precision syringe pump.
Perfusion was begun 20-24 hours after
the operation. The HPLC column was
MA-5ODS (4.6 x 150 mm), the mobile
phase was 0.1 M sodium citrate-acetate
buffer (pH = 3.5) containing 0.1 mM
EDTA and 17% methanol.
Perfusion solution:
The dialysate were collected for 25
minutes intervals, and were applied
directly onto the HPLC-ECD apparatus.
The ATX-III containing solution was 10
ug per ml Ringer. ATX-III was purified
from laboratory in Nagoya City
University (Hotta et al. 1980). DA and
its
metabolites,
DOPAC,
HVA
(homovanilic acid), NE, epinephrine,
and
HMPG
(4-hydroxy-3-methoxy
phenylethylene glycol) as well as 5 HT
and its metabolite, 5HIAA (5hydroxyindoleacetic
acid)
were
determined in the perfusate solution by
HPLC.
RESULTS
The perfusion of Ringer solution
achieved a steady state conditioned at
least after 50 minutes in brain striatum
microdialysis technique. Then the
concertration of DOPAC, HVA and 5
HIAA remained about 20% after 75
minutes perfusion with Ringer contaning
ATX-III (Fig. 1).
The contents of DA and serotonin
metabolites in the perfusate decreased
after injected with ATX-III into striatum
rat 5 months old (Fig. 2). DOPAC
concentration reduced from 9.3 ng into
4.4 ng/25 ul while HVA level that from
3.9 ng into 1.3 ng/25 ul. It was found
that 5HT metabolite as 5HIAA also
reduced markedly from 1.5 ng into 0.2
ng/25 ul. But DA content in the
perfusate was increased markedly from
1.1 pg into 9.9 pg/25 ul while 5 HT that
Majalah Kedokteran Andalas No. 2. Vol.23. Juli- Desember 1999
63
Microdialysis Study
from 2.6 pg into 7.2 pg/25 ul after 100
minutes injection with ATX-III (Fig.3)
The contents of ATX-III in nucleus
accumbence area that DOPAC, HVA
and 5HIAA decreased gradually into 60,
62 and 42% after 175 minutes injection
(Fig.4). Content of DOPAC reduced
from 0.5 ng into 0.34 ng/25 ul, HVA
from 0.33 to 0.3 ng/25 ul and 5HIAA
from 0.36 into 0.23 ng/25 ul after
injection with ATX-III into nucleus
accumbence (Fig.5). But content of DA
and serotonin in the perfuse increased
significantly after injection with Ringer
containing ATX-III solution. DA
concertration incrased from 1 pg into 1.5
pg/25 ul but 5HT level that almost three
times that from 3.1 to 8.9 pg/25 ul (Fig.
6).
The effects of ATX-III on
catabolisme of neurotransmitters showed
that DA metabolites, DOPAC, HVA and
MHPG gradually decreased as well as
that serotonin metabolite, 5HIAA (n=3)
Fig. 7 However release of DA, serotonin
and NE increased after ATX-III injection
into brain striatum rat in the same animal
experiment (Fig. 8).
DISCUSSION
A typical data showed that after at
least 50 minutes perfusion studies with a
physiologic solution, the steady state
conditioned was achieved in brain rat
striatum. Then DA and serotonin
catabolism decreased after direct
injection of ATX-III into the brain
ventricle. In different region of brain,
nucleus accumbence as a autonomic
nerve centre, also found a typical data
with decreasing of dopamine and
serotonin catabolism after ATX injection
(n=1 or 2).
Dopamin and serotonin catabolism
decreased since its metabolites markedly
reduced after direct injection of ATX
into striatum. However the NE level was
also found to increase markedly almost
in the same pattern with serotonin in the
striatum ATX injection. Therefora a
higher level of DA, serotonin and NE
might be caused by ATX-III stimulation
to the neurotransmitters release.
Effect of direct injection ATX-III
into brain striatum would increased
sympathomimetic activity of NE and
serotonin.
Content of DA in perfuse ATX
injection was found increased after 25
minute. In this perfusion study, DA level
in striatum befofe ATX injection as a
control animal are very low or
undetectable. It is necessary to
investigate the effects of ATX-III in high
dopaminergic activity of brain area such
as prefrontal cortex.
Dose of ATX-III during 25 minutes
perfusion was calculated 0.5 ug. In the
case of a higher ATX-III dose would be
able to produce massive symphatetis
discharge as their hypothesis by Takeya
et al. (1981). Eventhough 2.0 ug ATX
content after perfusion 100 min into
brain striatum was injection with a
slowly rate (2 ul/min).
Serotonin has a multiple actions, it
have induced contraction of pulmonary
arteries of bovine with a smooth muscle
receptor (frenken and Kaumann 1984)
Serotonin has been shown to elicit
vasorelaxation by an endotheliumdependent receptor mechanism (Leff et
al. 1987). Eventhough the lung was able
to rapidly remove serotonin from
circulation by a Na+_dependent
transport mechanism (Strum and Junod
Majalah Kedokteran Andalas No. 2. Vol.23. Juli- Desember 1999
64
Microdialysis Study
1987), the mechanism was in the rate
limiting strep.
DA has a specific vasodilator effect,
it has two sub-types of receptors in the
periphery (Goldberg and Kohli 1983).
The receptors was in the adventitial, the
adventitial border and the intimal leyer
of arteries (Amenta et al. 1990). The
majority of DA receptors in the rabbit
pulmonary artery are endothelial (Ricci
et al. 1992). Recently Boundy et al.
(1993) reported D3 dopamine receptor in
rat brain. DA was able to elicit alpha
adrenoceptor-mediated vasoconstriction
in pulmonary circulatin an anesthersized
dog (Shebuski et al. 1987).
Noradrenergic neurons of the rabbit
pulmonary artery are endowed with
presynaptic alpha-adrenoreceptor which
determine the rate of impulse flow (Endo
et al. 1977). Other researchers
(Greenberg et al. 1981) found that
intralobar pulmonary srteries and veins
in canine contract in response to norepinephrin. Rat pulmonary artery was
reported
content
both
alpha
adrenoceptors mediating contraction and
beta-1 and beta-2
adrenoceptors
mediating relaxation (O'Donnell and
Wanstall 1984).
In the conclusion of this study a
high level of serotonin, DA and NE after
direct injected ATX-III into brain
ventricle would played an important role
to produce a lung hemorrhage in animal
experiment.
It was necessary a further
investigation to study the effects of
direct injection of ATX-III in other brain
region. It was also to identify some
unknown substances in the perfusat
during microdialysis.
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