Document 9894

Journal of Cerebral Blood Flow and Metabolism
18:991-997 © 1998 The International Society of Cerebral Blood Flow and Metabolism
Published by Lippincott Williams & Wilkins, Philadelphia
Enhanced Poly(ADP-ribosyl)ation After Focal Ischemia in
Rat Brain
Tomoo Tokime, Kazuhiko Nozaki, Toshiyuki Sugino, Haruhiko Kikuchi, Nobuo Hashimoto,
and *Kunihiro Veda
Department of Neurosurgery, Faculty of Medicine, Kyoto University, Kyoto, Japan; and *Laboratory of Molecular Clinical
Chemistry, Institute for Clinical Research, Kyoto University, Kyoto, Japan
Summary: Nitric oxide from neuronal cells plays detrimental
roles in glutamate neurotoxicity and in focal brain ischemia,
Nitric oxide directly damages DNA, and breaks in the DNA
strands activate poly(ADP-ribose) polymerase (PARP), which
brings poly(ADP-ribosyl)ation of the nuclear proteins, The ex­
cessive activation of PARP is thought to cause depletion of
ATP and the energy failure resulting in cell death, To clarify
the involvement of poly(ADP-ribosyl)ation in ischemic insult,
we examined poly(ADP ribosyl)ation by immunohistochemical
methods and the protective effect of3-aminobenzamide, which
is a PARP inhibitor, on focal brain ischemia using an intralu­
minal permanent middle cerebral artery occlusion model in
rats, Poly(ADP ribosyl)ation was widely and markedly detected
2 hours after the ischemic insult in the cerebral cortex and
striatum in which infarction developed 24 hours later, The en­
hanced immunoreactivity of poly(ADP-ribose) gradually de­
creased, and 16 hours later, no immunoreactivity was detected,
Intraventricular administration of 3-aminobenzamide (1 to 30
mg/kg)30 minutes before the ischemic insult decreased infarc­
tion volume in a dose-dependent manner along with the immu­
nohistochemical reduction of poly(ADP-ribosyl)ation, Pretreat­
ment with 7-nitroindazole (25 mg/kg, intraperitoneally), a se­
lective neuronal nitric oxide synthetase inhibitor, partially
reduced poly(ADP-ribosyl)ation, These data suggest the in­
volvement of poly(ADP-ribosyl)ation in the development of
cerebral infarction, Key Words: Poly(ADP-ribosyl)ation­
Poly(ADP-ribose) polymerase-3-Aminobenzamide-Nitric
oxide-Focal ischemia-Rat
In cultured neurons, nitric oxide (NO) mediates glu­
tamate neurotoxicity, but in ischemic circumstances, NO
plays both beneficial and detrimental roles, and the effect
of nitric oxide synthetase (NOS) inhibitors in focal brain
ischemia has been controversiaL This is mainly because
NO is produced from various NOS isoforms (Iadecola et
aL, 1994), It has been shown that a specific neuronal
NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), is neuro­
protective in the rat focal brain ischemia model (Yoshida
et aL, 1994), and that nNOS knockout mice are resistant
to focal brain ischemia (Huang et aL, 1994), These data
suggest that NO produced from neurons is cytotoxic in
focal ischemia, The free radical NO reacts with super­
oxide anion and produces peroxynitrite, which is one of
the most powerful radicals, Peroxynitrite and NO di­
rectly damage DNA and enzymes involved in mitochon­
drial respiration (Wink et aL, 1991), Studies indicate that
one major pathway in which NO may exert toxicity is the
activation of poly(ADP-ribose) polymerase (PARP)
through damaging DNA, In neuronal cell cultures, NO
and glutamate are able to activate PARP through dam­
aging DNA (Cosi et aL, 1994; Zhang et aL, 1994, 1995),
Once activated, PARP catalyzes poly(ADP-ribosyl)ation,
the transfer of ADP ribose units from NAD to nuclear
proteins, The excessive activation of PARP causes deple­
tion of NAD and ATP, and leads to an energy failure,
Furthermore, the inhibition of PARP reduces the neuro­
toxicity of NO and glutamate (Cosi et aL, 1994; Zhang et
aL, 1994, 1995), These data indicate that the energy fail­
ure from excessive activation of PARP takes part in NO
and glutamate neurotoxicity,
The current study clarifies-by using an intraluminal
permanent middle cerebral artery (MCA) occlusion
model in rats-the immunohistochemical involvement of
poly(ADP ribosyl)ation and the neuroprotective effect of
3-aminobenzamide (3-ABA), which is a PARP inhibitor,
as they affect the development of cerebral infarction,
Received March 3, 1997; final revision received December 24, 1997;
accepted January 5, 1998,
Address correspondence and reprint requests to Dr. Tomoo Tokime,
Department of Neurosurgery, Faculty of Medicine, Kyoto University,
54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-01, Japan,
Abbreviations used: 3-ABA, 3-aminobenzamide; MeA, middle ce­
rebral artery; 7 -NI, 7 -nitroindazole; NO, nitric oxide; NOS, nitric oxide
synthetase; nNOS, neuronal nitric oxide synthetase; PARP, poly(ADP­
ribose) polymerase,
Induction of focal brain ischemia
Male Sprague-Dawley rats (Shimizu Laboratory Supplies
Co., Ltd., Kyoto, Japan) weighing 280 to 320 g were used.
After overnight fasting, rats were anesthetized with 4.0% halo­
thane and maintained with 1 .0% halothane in a gas mixture of
30% oxygen170% nitrous gas using a face mask. The rectal
temperature was maintained at 37.5 ± 0.5°C during surgery
with a heating lamp and a heating pad connected to a rectal
thermistor. The right femoral artery was cannulated to record
blood pressure and to obtain arterial blood samples. Focal brain
ischemia was induced under spontaneous respiration according
to a method of intraluminal MCA occlusion with slight modi­
fication (Longa et a!., 1 989). Briefly, the right common carotid
artery, external carotid artery, and internal carotid artery were
isolated through a midline cervical skin incision under a mi­
croscope. Approximately 1 8 mm of 4-0 nylon monofilament,
which was blunted by coating with silicone, was advanced from
the lumen of the external carotid artery into the internal carotid
artery to block the origin of the right MCA. Rats were allowed
to survive for the indicated period before killing. Control ani­
mals were operated without MCA occlusion.
Pretreatment with 3-aminobenzamide
weakly reactive with short oligomers. The specificity was con­
firmed by the disappearance of the immunostaining by preab­
sorption of the antiserum with purified poly(ADP-ribose) or by
pretreatment of tissue sections with poly(ADP-ribose)­
degrading enzymes. The sections were washed with phosphate­
buffered saline, and then incubated with biotinylated anti-rabbit
IgG antibody at 1 :200 dilution for 2 hours and with an avidin­
biotin complex (ABC kit from Vector) for 60 minutes. Peroxi­
dase was demonstrated with a DAB substrate kit (Funakoshi,
Tokyo, Japan). Negative control sections received identical
treatment except for the primary antibody. We also used the
commercially available polyclonal anti-poly(ADP-ribose) anti­
body prepared from guinea pig serum (Trevigen, Gaithersburg,
MD, U.S.A.) to check interantibody differences in immunore­
activity. For evaluation of morphologic change, contiguous
sections were stained with cresyl violet.
Measurement of infarction volume
To examine the effect of intraventricular administration of
3-ABA30 minutes before the ischemia on the development of
cerebral infarction, rats were killed 24 hours later after isch­
emic insult for 2,3,5-triphenyltetrazolium chloride (nacalai
tesque) staining (Bederson et aI., 1 986) to measure infarction
volume. Twenty-seven rats were used (n
7, 4, 6, 6, 4 for
vehicle and doses of 1 ,3, 1 0, and30 mg/kg, respectively). Rats
were decapitated, the brains were removed immediately, and
seven coronal slices 2, 4, 6, 8, 1 0, 1 2, and 1 4 mm distal from
the frontal pole were dissected using a brain slicer. Infarction
areas were calculated for each coronal slice by NIH Image (ver.
1 .56), and infarction volumes were determined by numeric in­
tegration of infarction areas. Data were presented as mean ± SD
and statistically analyzed using analysis of variance followed
by post hoc Bonferroni test using Stat View II (Abacus Con­
cepta, Berkeley, CA, U.S.A.), and when P is less than 0.05,
differences were considered significant.
and 7-nitroindazole
To examine the effect of 3-ABA (nacalai tesque, Kyoto,
Japan), a PARP inhibitor, on brain ischemia,3-ABA (0, I, 3,
1 0,30 mg/kg) was administered into the right lateral ventricle
stereotactically 30 minutes before the ischemic insult under
general anesthesia (described earlier). In a preliminary experi­
ment, a dose of I to 1 00 mg/kg of 3-ABA was administered
intraperitoneally30 minutes before the ischemic insult with no
reduction of infarction volume. Therefore, we used intraven­
tricular injection of3-ABA. The heads of rats were secured in
a stereotactic frame, and a 26-gauge needle was inserted into
the right lateral ventricle. Coordinates were 1 .5 mm lateral, 0.8
mm posterior, and3.3 mm ventral from the dural surface using
bregma as a landmark. Thirty microliters of each drug solution
were injected over 5 minutes. The 3-ABA was dissolved in
distilled water, and pH was adjusted to 7.0. To examine the
involvement of NO from nNOS in the induction of poly(ADP­
ribosy\)ation, 7-NI (25 mg/kg) (Lancaster Co., Edinburg, U.K.)
was administered intraperitoneally30 minutes before the isch­
emic insult. The 7-NI was suspended in peanut oil (nakalai
tesque, Kyoto, Japan).
Immunohistochemistry of poly(ADP-ribose)
Two hours after the intraluminal right MeA occlusion,
neuronal damage was detected only in the medial part of
Tissue preparation and immunohistochemistry
To clarify the temporal and spatial distribution of poly(ADP­
ribosyl)ation after focal brain ischemia, rats were transcardially
perfused with 4% paraformaldehyde in phosphate-buffered sa­
line (pH 7.4) at 1 , 2, 4, 8, 16, and 24 hours after the induction
of ischemia (n
3 at each time point). Control animals (n
3) were killed 8 hours after operation. To examine the effect of
3-ABA (0, 1 ,3, 1 0,30 mg/kg) and 7-NI (25 mg/kg) on poly­
(ADP-ribosyl)ation after ischemia, rats (n
3 for each dose)
were perfused as described earlier at 2 hours after ischemia.
The brains were rapidly removed and cryoprotected in 25%
sucrose in phosphate-buffered saline overnight at 40c. Frozen
coronal sections (50-fLm thickness) were prepared. After
quenching endogenous peroxidase in 2% H202 in 60% metha­
nol and blocking with 5% goat serum, sections were incubated
overnight at 4°C with polyclonal antibody against poly(ADP­
ribose) (Ikai et aI., 1 980). This antibody was a polyclonal an­
tibody raised in rabbits against purified poly(ADP-ribose) (av­
erage chain length, 24); it was most reactive with polymers
having the chain length of about 25 ADP-ribose units and
J Cereb Blood Flow Metab. Vol. 18. No.9. 1998
FIG. 1. Schematic illustration of positive immunoreactivity for
poly(ADP-ribosyl)ation and neuronal degeneration. Top: Tempo­
ral profile and distribution of immunoreactive cells for poly(ADP­
ribosyl)ation. One dot indicates approximately five strongly im­
munopositive cells. The photographs were taken as shown in Fig.
2 at the site of squares 1 through 3. Square 1 is the center of
ischemic core in the cortex. Square 2 is periinfarct area (ischemic
penumbra). Square 3 is the lateral part of striatum (ischemic
core). Areas 1, 2, and 3 are on the same section, 7.2 mm anterior
to interaural line. Bottom: Temporal profile and distribution of
neuronal damage. Neuronal degeneration was evaluated by
Nissl stain. Bright shadow indicates about 30% of neurodegen­
eration. Dark shadow indicates more than 70% of neurodegen­
FIG. 2. Representative microphotographs of poly(ADP-ribose) immunohistochemical study at 0 (A-C), 2 (D-F), 8 (G-I) and 24 (J-L) 2
hours after ischemic insult. Figs. 2A, D, G, and J were taken at the site of square 1. Figs. 28, E, H, and K, and C, F, I, and L were taken
at the site of squares 2 and 3, respectively. No immunoreactive cells were observed 0 hours after ischemic insult (A-C). Poly(ADP­
ribosyl)ation was found throughout the ischemic area 2 hours after ischemic insult. Immunoreactivity was found mainly in nuclei (D-F).
Eight hours after ischemic insult, the positive nuclei appeared larger and rounder (G-I). No positive cells were found 24 hours after
ischemic insult (J-L). All of the photographs were taken under the same magnification. The bar in Fig. 2L indicates 20 IJm.
right striatum in cresyl violet staining. Neuronal damages
gradually expanded thereafter, and 24 hours later a dam­
aged area expanded to most of the right striatum and
right frontoparietal cortex (Fig. 1).
We used two polyclonal antibodies, and temporal and
spatial distribution of poly(ADP-ribosyl)ation was essen-
tially the same. The main difference between the immu­
noreactivities in two antibodies was the intracellular dis­
tribution. The antibody from by Ikai and others produced
strong immunoreactivity, mainly in the nuclei with faint
staining in the cytoplasm (Figs. 2 and 3), but the anti­
body from Trevigen produced diffuse immunoreactivity,
J Cereb Blood Flow Metab. Vol. 18. No.9. 1998
or frontoparietal cortex (Figs. 1 and 2A through C). Faint
poly(ADP-ribose) immunoreactivity was detected only
in neurons of the piriform cortex and cingulate gyrus.
One hour after ischemic insult, poly(ADP-ribosyl)ation
was weakly detected in the medial and lateral striatum
and in the frontoparietal cortex (data not shown). Two
hours after ischemic insult, poly(ADP-ribosyl)ation
peaked in the lateral striatum and throughout the fronto­
parietal cortex (Fig. 2D through F). We confirmed that
the positive cells were mostly neurons by using morpho­
logic study and a double staining method (data not
shown). Among intracellular organelles, nuclei were
stained prominently with occasional faint staining in the
cytoplasm (Fig. 3A). In the medial part of striatum, in
which neurodegeneration was severe, the staining was
less dense. Ischemic penumbra in the cortex, which is
indicated as square 2 in Fig. 1, showed moderate immu­
noreactivity. The piriform cortex, in which neuronal de­
generation could not be found 24 hours later in this
model, also showed moderate immunoreactivity. Four
hours after ischemic insult, the number of positive cells
gradually decreased. Eight hours after ischemic insult
(Fig. 2G through I), the positive cells decreased rapidly
in the striatum, and the positive cells were prominent
only in the fifth layer of the cortex, although the piriform
cortex was stained moderately. At this time, the staining
showed a larger and rounder shape, probably because of
degeneration of nuclei (Fig. 2G). Sixteen hours and 24
hours after intraluminal MCA occlusion, the immunore­
activity of poly(ADP-ribose) almost completely disap­
peared (Figs. 1 and 2J through L). Contralateral hemi­
sphere showed no poly(ADP-ribose) immunoreactivity
during the ischemic period, except faint staining in the
piriform cortex and cingulate gyrus. Negative control
without primary antibody did not show poly(ADP­
ribose) immunoreactivity, either.
The effect of 3-aminobenzamide and 7-nitroindazole
on poly(ADP-ribosyl)ation
FIG. 3. Representative photomicrographs of poly(ADP-ribose)
immunohistochemical study at the area of square 1 (indicated in
Fig. 1) 2 hours after ischemic insult in animals that received in­
traventricular injection of vehicle (A), intraventricular injection of
10 mg/kg of 3-aminobenzamide (3-ABA) (8), and intraperitoneal
injection of 25 mg/kg of 7-nitroindazole (7-NI) (C) 30 minutes
before ischemic insult. In animals that received vehicles, poly­
(ADP-ribose) immunoreactivity was the same as in animals that
received only ischemic insult. Intraventricular administration of 10
mg/kg of 3-ABA markedly reduced the immunoreactivity (8). In­
traperitoneal injection of 25 mg/kg of 7-NI moderately reduced
the immunoreactivity (C). The bar in C indicates 10 �m.
both in the nuclei and cytoplasm (see Sugino et aI, 1997).
In the current study, we demonstrated the results ob­
tained using the former, since the antibody from Trevi­
gen produced higher background than the polyclonal an­
tibody by Ikai and colleagues.
In the control group and 0 hours after ischemic insult,
no poly(ADP-ribosyl)ation was detected in the striatum
J Cereb Blood Flow Metab. Vol. 18. No.9, 1998
Intraventricular administration of 3-ABA (0, I, 3, 10,
30 mg/kg) 30 minutes before the ischemic insult reduced
poly(ADP-ribose) immunoreactivity 2 hours after isch­
emia in a dose-dependent manner, and the maximum
reduction of the poly(ADP-ribose) immunoreactivity
was obtained with a dose of 10 mg/kg (Fig. 3A and B).
The reduction of immunoreactivity was more prominent
in the cortex than in the striatum (data not shown). In­
traperitoneal administration of 25 mg/kg of 7-NI moder­
ately reduced poly(ADP-ribose) immunoreactivity 2
hours after ischemia (Fig. 3C), but less effectively than
The effect of 3-aminobenzamide on
infarction volume
Twenty-four of 27 rats were survived for 24 hours
after 3-ABA treatment. Mortality rates were 5/7, 3/4, 6/6,
tained at a dose of 10 mg/kg (Figs. 4 and 5). The reduc­
tion of infarction volume was more prominent in the
cortex than in the striatum. Systemic blood pressure, ar­
terial gas analysis (pH, Paz, Pcoz), hematocrit, and blood
glucose were not significantly different between groups
5 minutes before the ischemia (Table 1).
Poly(ADP-ribose) polymerase is activated by DNA
breakage, resulting in the addition of up to 100 ADP­
ribose groups to acceptors such as histone and PARP
itself. After limited damages of DNA, poly(ADP­
ribosyl)ation plays a critical role in DNA repair (de Mar­
cia and de Marcia, 1994). However, when massive dam­
age of DNA occurs, the associated extensive activation
of PARP is thought to lead to depletion of NAD, which
is the donor of the ADP-ribose group. Also, ATP is
depleted in efforts to resynthesize NAD, resulting in cell
death. Zhang and associates (1994) showed that NO ac­
tivated PARP and that the inhibition of PARP rescued
from NO- and N-methyl-D-aspartate-mediated neurotox­
icity in a cortical culture. Cosi and colleagues (1994)
showed by immunohistochemical study that glutamate
induced poly(ADP-ribosyl)ation in cerebellar granule
cells. They also showed that inhibitors of PARP reduced
glutamate neurotoxicity. Also, in other cell lines, the in­
hibition of PARP has been shown to induce protective
effects on cytotoxicity by several stimuli, including NO
(Burkart et aI., 1995; Heller et aI., 1995; Kuo et a1., 1996;
Nosseri et aI., 1992; Radons et aI., 1994; Zingarelli et aI.,
1996). These results strongly suggest the involvement of
poly(ADP-ribosyl)ation in the process of cell death. An­
other mechanism of cell death by poly(ADP-ribosyl)ation
FIG. 4.
Total, cortical, and striatal infarction volumes 24 hours
after intraluminal, permanent right middle cerebral artery (MCA)
occlusion in animals that received 0 (vehicle), 1, 3, 10, and 30
mg/kg of intraventricular administration of 3-ABA 30 minutes be­
fore ischemic insult. The 3-ABA reduced total, cortical, and stria­
tal infarction volume dose dependently. The maximum reduction
is obtained at a dose of 10 mg/kg. Data are presented as means
± SO, and P < 0.05,
P < 0.0 1 when compared with vehicle.
6/6, and 4/4 for vehicle and doses of 1, 3, 10, and 30
mg/kg of 3-ABA, respectively. Intraventricular adminis­
tration of 3-ABA (0, 1, 3, 10, and 30 mg/kg) 30 minutes
before the ischemic insult significantly reduced infarc­
tion volume 24 hours after the ischemia in a dose­
dependent manner, and the maximum effect was ob-
... cortical, control
-&- cortical, lOmg!kg
.....- striatal, control
--e- striatal,
Distance from frontal pole (mm)
FIG. 5.
Cortical (square) and striatal (circle) infarction areas at
each coronal section (2, 4, 6, 8, 10, 12, and 14 mm from frontal
pole) in animals that received vehicle (control; closed) and 10
mg/kg of intraventricular administration of 3-ABA (open) 30 min­
utes before ischemic insult. Data are presented as means ± SO,
and P < 0.05,
P < 0.0 1 when compared with vehicle.
J Cereb Blood Flow Metab. Vol. 18, No.9, 1998
TABLE 1. Physiologic parameters before intraluminal middle cerebral artery occlusion under
halothane anesthesia
(mm Hg)
I mg/kg
3 mg/kg
IO mg/kg
30 mg/kg
MABP (mean arterial blood pressure), arterial blood gas (pH, Po" Peo2), Hct (hematocrit), and blood glucose
were measured 5 minutes before middle cerebral artery occlusion. Data are presented as mean±SD.
other than energy failure was proposed by Nosseri and
coworkers (1992). They speculated that poly(ADP­
ribosyl)ation may represent a quantity of DNA damages,
and some intracellular signal transduction mechanisms
for cell death may exist after poly(ADP-ribosyl)ation.
The current study showed that poly(ADP-ribosyl)ation
was detected 1 hour and peaked 2 hours after focal brain
ischemia model, where infarction developed several
hours later. These findings indicate that PARP is acti­
vated in vivo in the early phase of the development of
infarction. Because PARP recognizes the site of DNA
single-strand breaks but not the site of double-strand
breaks, poly(ADP-ribosyl)ation in the focal cerebral
ischemia may reflect early damages of single-strand
DNA in ischemic regions.
When we used the antibody by Ikai and colleagues,
poly(ADP-ribose) immunoreactivity was mainly located
in the nucleus of neurons, where DNA damages and
repairs should occur. A few neurons in severe ischemic
regions showed weak poly(ADP-ribose) immunoreactiv­
ity in the cytoplasm, along with strong poly(ADP-ribose)
immunoreactivity in the nucleus. The commercially
available polyclonal antibody (Trevigen) produced dif­
fuse, positive staining both in the nucleus and cytoplasm.
Further study is needed to clarify whether activation of
PARP and poly(ADP-ribosyl)ation will occur in the cy­
toplasm, as well as the locus of poly(ADP-ribosyl)ation
in the cytoplasm.
The current study also demonstrates that 3-ABA,
which is a PARP inhibitor, reduced mortality rate and
also reduced infarction volume dose dependently, along
with the reduction of poly(ADP-ribosyl)ation in perma­
nent MCA occlusion model in rats. The maximum re­
duction in infarction volume was obtained by 40% at a
dose of 10 mg/kg. In this dosage, poly(ADP­
ribosyl)ation did not disappear completely. During the
preparation of this manuscript, Eliasson et aI. (1997)
showed that genetic disruption of PARP provides pro­
found protection against glutamate-NO-mediated isch­
emic insults in vitro and major decreases in infarction
volume after reversible MCA occlusion. They have dem­
onstrated 80% reduction in infarction volume in PARP
knockout mice. The differences in the neuroprotective
J Cereb Blood Flow Metab, Vol. 18, No.9, 1998
effect of PARP inhibition in these two experiments may
reflect the difference in the ischemia model (permanent
versus transient) and the drug delivery and bioavailabil­
It has been recently shown that NO and glutamate are
able to activate PARP through damaging DNA in neu­
ronal cell culture (Cosi et aI., 1994; Zhang et aI., 1994,
1995). We have shown in the current study that focal
brain ischemia induced poly(ADP-ribosyl)ation, and that
7-NI, a specific nNOS inhibitor, reduced poly(ADP­
ribosyl)ation in vivo. Because 7-NI could not block poly­
(ADP-ribosyl)ation completely, PARP may be activated
through NO, as well as other stimuli, during focal brain
The current study demonstrates the strong involve­
ment of poly(ADP-ribosyl)ation in the development of
cerebral infarction in focal ischemia model, and implies
a new therapeutic modality for ischemic brain injury
through the inhibition of poly(ADP-ribosyl)ation.
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