Journal of Cerebral Blood Flow and Metabolism 17:1137-1142 © 1997 The International Society of Cerebral Blood Flow and Metabolism Published by Lippincott-Raven Publishers, Philadelphia

Neuroprotective Effects of Inhibiting Poly(ADP-Ribose)

Synthetase on Focal Cerebral Ischemia in Rats

Kazushi Takahashi, Joel H. Greenberg, *Paul Jackson, *Keith Maclin, and *Jie Zhang

Cerebrovascular Research Center, Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia,

Pennsylvania; and *Guilford Pharmaceuticals, Inc., Baltimore, Maryland, U.S.A.

Summary: Poly(adenosine 5' -diphosphoribose) synthetase (PARS) has been described as an important candidate for me­diation of neurotoxicity by nitric oxide. In the current study, we demonstrate for the first time that in vivo administration of a potent PARS inhibitor, 3,4-dihydro 5-[4-1(1-piperidinyl) bu­toxy ]-1 (2H)-isoquinolinone, leads to a significant reduction of infarct volume in a focal cerebral ischemia model in the rat. Focal cerebral ischemia was produced by cauterization of the right distal middle cerebral artery (MCA) with bilateral tem­porary common carotid artery occlusion for 90 minutes. 3,4-Dihydro 5[4-( I-piperidinyl) butoxy ]-1 (2H)-isoquinolinone was dissolved in dimeth�1 sulfoxide and injected intraperitoneally. Animals were treated 2 hours before MCA occlusion (control, n = 14; 5 mg/kg, n = 7; 10 mg/kg, n = 7; 20 mg/kg, n = 7; 40 mg/kg, n = 7), and 2 hours after MCA occlusion (same

Nitric oxide (NO) originally was identified as the en­

dothelium-derived relaxing factor (Ignarro et aI., 1987;

Palmer et aI., 1987). It was subsequently found to be a

messenger molecule in the nervous system (Garthwaite

et aI., 1988; Bredt and Snyder, 1989). Excessive NO

production has been implicated in playing a significant

role in the N-methyl-o-aspartate (NMDA) receptor­

mediated neurotoxicity of the excitatory amino acid glu­

tamate in cerebral ischemia (Dawson et aI., 1991, 1993).

NMDA receptor activation causes an increase in intra­

cellular calcium concentration, leading to an activation

of neuronal NO synthase. One of the best-established

candidates for mediator of neurotoxicity by NO is poly-

Received June 5, 1997; final revision received July I, 1997; accepted July 16, 1997.

Supported by National Institutes of Health Grant NS32017 and Guilford Pharmaceuticals, Inc., Baltimore, Maryland, U.S.A.

Address correspondence and reprint requests to Dr. Joel H. Greenberg, Cerebrovascular Research Center, 429 Johnson Pavilion, 3610 Hamilton Walk, University of Pennsylvania, Philadelphia, PA 19104-6063, U.S.A.

Abbreviations used: CCA, common carotid artery; MCA, middle cerebral artery; NMDA, N-methyl-D-aspartate; NO, nitric oxide; PARS, poly(adenosine 5' -diphosphoribose) synthetase.

1137

doses as before treatment). Twenty-four hours after MCA oc­clusion, the total infarct volume was measured using 2,3,5-triphenyltetrazolium chloride. Inhibition of PARS leads to a significant decrease in the damaged volume in the 5 mg/kg­treated group (106.7 ± 23.2 mm3; mean ± SD, P < 0.002), the 10 mg/kg-treated group (76.4 ± 16.8 mm3, P < 0.001), and the 20 mg/kg-treated group (110.2 ± 42.0 mm3, P < 0.02) com­pared with the control group (165.2 ± 34.0 mm3). The substan­tial reduction in infarct volume indicates that the activation of PARS may play an important role in the pathogenesis of brain damage in cerebral ischemia through intracellular energy depletion. Key Words: Cerebral ischemia-Middle cerebral artery occlusion-Neuroprotection-Nitric oxide-Poly(ADP­ribose) polymerase-Rat.

(adenosine 5'-diphosphoribose) synthetase (PARS). The

NO-damaged DNA activates PARS, which can rapidly

delete NAD (the source of ADP-ribose) and lead to cell

death (Zhang et aI., 1994).

Also called PARP [for poly(ADP-ribose) polymerase

(EC 2.4.2.30)], PARS is a tightly bound chromosomal

enzyme located in the nuclei of cells of various organs

including brain (Ikai et aI., 1980; Ogura et aI., 1990). PARS plays a physiologic role in the repair of strand

breaks in DNA (Satoh and Lindahl, 1992). Once acti­

vated by damaged DNA fragments, PARS catalyzes the

attachment of ADP-ribose units to nuclear proteins, in­

cluding histones and PARS itself. The PARS activation

enhances DNA repair by relaxing chromosomal structure

through poly(ADP-ribosyl)ation of histones and other

nuclear proteins. The extensive activation of PARS,

however, can rapidly lead to cell death through depletion

of energy stores, since four molecules of ATP are con­

sumed in NAD (the source of ADP-ribose) regeneration

(Berger, 1985). Previously published data show that a

series of PARS inhibitors protects various cell cultures

from NO- and glutamate-mediated neurotoxicity (Miller

et aI., 1993; Cosi et aI., 1994; Radons et aI., 1994) and

1138 K. TAKAHASHI ET AL.

reactive oxygen radical-induced injury (Thies and Autor,

1991; Aalto and Raivio, 1993). Despite many in vitro studies using inhibitors of PARS, there have been no

investigation on the effect of in vivo administration of

PARS inhibitors in cerebral ischemia.

A new series of highly potent inhibitors of PARS, the

dihydroisoquinolinones, recently has been reported (Suto

et aI., 1993). 3,4-Dihydro 5-[4-(l-piperidinyl) butoxy] ­

I (2H)-isoquinolinone is one member in this potent series

of PARS inhil;litors. 3,4-Dihydro 5-[ 4-(l-piperidinyl) bu­

toxy ]-1 (2H)-isoquinolinone is mimicking nicotinamide

to competitively inhibit PARS at the NAD binding site.

The current study examines the effect of in vivo admin­

istration of 3,4-dihydro 5-[4-(l-piperidinyl) butoxy]-

1 (2H)-isoquinolinone on focal cerebral ischemia pro­

duced by middle cerebral artery (MeA) occlusion in the

rat.

MATERIALS AND METHODS

Animal preparation All procedures performed on animals were approved by the

University Institutional Animal Care and Use Committee of the University of Pennsylvania. A total of 42 male Long-Evans rats (weights 230 to 340 g) obtained from Charles River Laboratory (Wilmington, MA, U.S.A.) were used in this study. The ani­mals were faste� overnight with free access to water before the surgical preparation. They were anesthetized with halothane (4% for induction and 0.8% to 1.2% for surgical procedure) in a mixture of 70% nitrous oxide and 30% oxygen. The body temperature was monitored by a rectal probe and maintained at 37.SO ± 0.5°C with a heating blanket regulated by a homeo­thermic blanket control unit (Harvard Apparatus Limited, Kent, U.K.). A catheter (PE-50) was placed into the tail artery, and arterial pressure was continuously monitored and recorded on a Grass polygraph recorder (Model 7D, Grass Instruments, Quincy, MA, U.S.A.). Samples for blood gas analysis (arterial pH, Pa02 and Pac02) also were taken from the tail artery cath­eter and measured with a blood gas analyzer (ABL 30, Radi­ometer, Copenhagen, Denmark).

Laser Doppler flowmetry The head was positioned in a stereotaxic frame, and a right

parietal incision between the right lateral canthus and the ex­ternal auditory meatus was made. Using a dental drill con­stantly cooled with saline, a 3-mm burr hole was prepared over the cortex supplied by the right MCA, 4 mm lateral to the sagittal suture and 5 mm caudal to the coronal suture. The dura mater and a thin inner bone layer were kept, with care being taken to position the probe over a tissue area devoid of large blood vessels. The flow probe (tip diameter of 1 mm, fiber separation of 0.25 mm) was lowered to the bottom of the cra­nial burr hole using a micromanipulator. The probe was held stationary by a probe holder secured to the skull with dental cement. The microvascular blood flow in the right parietal cortex was continuously monitored with a laser Doppler flow­meter (FloLab, Moor, Devon, U.K.; and PeriFlux 4001, Perimed, Stockholm, Sweden).

Focal cerebral ischemia Focal cerebral ischemia was produced by cauterization of the

distal portion of the right MCA with bilateral temporary com-

J Cereb Blood Flow Metab. Vol. 17. No. 11. 1997

mon carotid artery (CCA) occlusion (Chen et aI., 1986; Liu et aI., 1989). Bilateral CCA were isolated, and loops made from polyethylene catheter (PE-lO) were carefully passed around the CCA for later remote occlusion. The incision made previously for placement of the laser Doppler probe was extended to allow observation of the rostral end of the zygomatic arch at the point of fusion to the squamous bone. A burr hole was made in the parietal bone 2 to 3 mm rostral to the fusion point using a dental drill, and the dura mater overlying the MCA was cut. The MCA distal to its crossing with the inferior cerebral vein was lifted by a fine stainless steel hook attached to a micromanipulator, and after bilateral CCA occlusion, the MCA was cauterized with an electrocoagulator. The burr hole was covered with a small piece of Gelform, and the wound was sutured to maintain the brain temperature. After 90 minutes of occlusion, the carotid loops were released, the tail arterial catheter was removed, and all of the wounds were sutured. Gentamicin sulfate (10 mg/mL; So-10Pak Laboratories, IL, U.S.A.) was topically applied to the wounds to prevent infection. The anesthesia was discontinued, and the animal was returned to its cage after awakening. At the dose we used in the study, 3,4-dihydro 5-[4-(l-piperidinyl) bu­toxyJ-l(2H)-isoquinolinone had no apparent effects on the rats in terms of mortality or recovery from anesthesia. No animals died during the study, and all rats awakened from the halothane anesthesia within 20 minutes after halothane was discontinued. Water and food were allowed ad libitum.

Therapeutic protocol 3 , 4 - D i h y d r o 5 - [ 4 - ( I- p i p e r i d i n y l ) b u t o x y ] - 1 ( 2 H ) ­

isoquinolinone was dissolved in 100% dimethyl sulfoxide us­ing a sonicator and injected intraperitoneally. Animals were treated with 3,4-dihydro 5-[ 4-( l -piperidinyl) butoxy ]-1 (2H)­isoquinolinone 2 hours before MCA occlusion (5 mg/kg, n =

7; 10 mg/kg, n = 7; 20 mg/kg, n = 7; 40 mg/kg, n = 7) and 2 hours after MCA occlusion (same doses as before treatment) in a volume of 1.28 mL of dimethyl sulfoxide per kilogram of body weight per injection. This dosing schedule was used to keep the volume of dimethyl sulfoxide constant across groups. Fourteen animals were injected similarly with equivalent vol­umes of the vehicle in the same schedule. These studies were designed to demonstrate the principle of in vivo PARS inhibi­tion for reducing neuronal damage during ischemia. Conse­quently, the PARS inhibitor was administered both before and after the start of ischemia so as to maximize the inhibition during the early ischemic period.

Morphometric measurement of damaged volume Twenty-four hours after the MCA occlusion, the rats were

killed with an intraperitoneal injection of pentobarbital sodium (150 mg/kg). The brain was carefully removed from the skull and cooled in ice-cold artificial CSF for 5 minutes and then sectioned in the coronal plane at 2-mm intervals using a rodent brain matrix (RBM-4000C, ASI Instruments, Warren, MI, U.S.A.) The brain slices were incubated in phosphate-buffered saline containing 2% 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, MO, U.S.A.) at 37 OC for 10 minutes. Color slides were obtained from the posterior surface of the stained slices and were used to determine the damaged area at each cross-sectional level using a computer-based image analyzer (NIH Image version 1.59). To avoid artifacts caused by edema, the damaged area was calculated by subtracting the area of the normal tissue in the hemisphere ipsilateral to the stroke from the area of the hemisphere contralateral to the stroke (Swanson et aI., 1990). The total volume of infarction was calculated by summation of the damaged volume of the brain slices.

NEUROPROTECTIVE EFFECTS OF INHIBITING POLY(ADP-RIBOSE) SYNTHETASE //39

Statistical analysis The data are expressed as mean ± SD. The significance of

differences between groups was determined using an analysis of variance followed by probing for the source of the difference using a Student's t test in which a Bonferroni correction was made for multiple comparisons.

RESULTS

Physiologic variables

The values of arterial blood gases (Pao2, Paco2, and

pH) were within the physiologic range in the control and

treated groups with no significant difference� in these

parameters among the five groups (Table 1). There were

no significant differences in mean arterial blood pressure

before MCA and CCA occlusion among the five groups.

Although mean arterial blood pressure was significantly

elevated after occlusion in all five groups, there were no

significant differences in mean arterial blood pressure

during the occlusion period among the groups (Table 1).

Effects of the right middle cerebral artery and

bilateral common carotid artery occlusion on

cerebral blood flow

Since the blood flow values obtained from the laser

Doppler are in arbitrary units, only percent changes from

the baseline (before occlusion) are reported. Right MCA

and bilateral CCl\. occlusion produced a significant de­

crease in relative blood flow in the right parietal cortex to

20.8 ± 7.7% of the baseline in the control group (n = 5),

18.7 ± 7.4% in the 5 mg/kg-treated group (n = 7), 21.4

± 7.7% in the 10 mglkg-treated group (n = 7), and 19.3 ± 11.2% in the 40 mglkg-treated group (n = 7). There

were no significant differences in the blood flow re­

sponse to occlusion among the four groups. In addition,

blood flow showed no significant changes throughout the

entire occlusion period in any group.

Effects of PARS inhibition on focal cerebral

ischemia

The cauterization of the distal portion of the right

MCA with bilateral temporary CCA occlusion consis­

tently produced a well-recognized cortical infarct in the

right MCA territory of each animal. There was an ap­

parent uniformity in the distribution of the damaged area

as measured by 2,3,5-triphenyltetrazolium chloride stain­

ing in each group (Fig. 1). Inhibition of PARS leads to a

significant decrease in the damaged volume in the 5 mg/

kg-treated group (106.7 ± 23.2 mm3, P < 0.002), the 10

mglkg-treated group (76.4 ± 16.8 mm3, P < 0.001), and

the 20 mglkg-treated group (110.2 ± 42.0 mm3, P < 0.02) compared with the control group (165.2 ± 34.0

mm3). However, there was no significant difference be­

tween the control and the 40 mglkg-treated group (135.6

± 44.8 mm3) (Fig. 2).

DISCUSSION

In the current study, we demonstrate for the first time

that in vivo administration of a PARS inhibitor leads to a

significant reduction of infarct volume in a focal cerebral

ischemia model in the rat. This suggests that the activa­

tion of PARS may play an important role in the patho­

genesis of brain damage in cerebral ischemia through

intracellular energy depletion.

In our focal ischemia model, the ligation of the right

distal MCA and bilateral temporary CCA occlusion for

90 minutes produced a large and well-recognized infarc­

tion. It has been shown that the ligation of the right distal

MCA only (without CCA occlusion) does not produce a

cortical infarction in Long-Evans rats (Chen et ai., 1986).

When both CCA also are temporarily occluded, as in the

current study, the infarct volume becomes 188 ± 18 mm3

(means ± SEM) (Liu et ai., 1989), and when both the

MCA and the CCA are reopened after 90 minutes of

occlusion, the volume of infarction is comparable (171 ± 16 mm3) (Yip et aI., 1991). These values are similar to

that observed in our study. After release of the carotid

occlusions, we observed a good recovery of blood flow

(sometimes hyperemia) in the right MCA territory of all

animals (data not shown). Reperfusion of the ischemic

tissue results in the formation of NO and peroxynitrite, in

TABLE 1. Physiologic variables

MABP (mm Hg)

Pao2 Paco2 pH Steady state* Ischemiat

Control (n = 14) 125 ± 21 38.6 ± 4.6 7.33 ± 0.05 79 ± 14 91 ± 13:1: 5 mglkg-treated group Cn = 7) 126 ± 20 38.0 ± 2.8 7.36 ± 0.02 78 ± 5 91 ± 12:1: 10 mglkg-treated group (n = 7) 125 ± 16 39.3 ± 5.2 7.34 ± 0.03 80±9 90 ± 14§ 20 mglkg-treated group Cn = 7) 122± 14 41.3 ± 2.8 7.35 ± 0.03 79 ± 10 91 ± 12:j: 40 mglkg-treated group Cn = 7) 137 ± 17 39.5 ± 4.7 7.33 ± 0.04 78 ± 4 88 ± 12§

Values are expressed as mean ± SD. Arterial blood samples were obtained 30 minutes after MCA occlusion. There was no significant difference among any of the groups in any physiologic parameter.

* After completion of the surgical preparation, just before occlusion. t Average MABP during occlusion. :I: Significantly different from the steady state value, P < 0.0\. § Significantly different from the steady state value, P < 0.05.

J Cereb Blood Flow Metab, Vol. 17, No. II, 1997

1140 K. TAKAHASHI ET AL.

--+-- 10 mg/kg-treated group (n=7)

1;:- Coo,,,, ("�'4) -­

'f 20 j J� I

S 15

co

� CO

"0 10 Q) OJ co

E co o 5

o

-2 0 2 4 6 8 10 12 14 16

Distance from interauralline (mm) FIG. 1. The distribution of the cross-sectional infarct area at rep­resentative levels along the rostrocaudal axis as measured from the interaural line in nontreated animals and in animals treated with 10 mg/kg of 3,4-dihydro 5-[4-(1-piperidinyl) butoxyj-1 (2H)­isoquinolinone. The area of damage is expressed as mean ± SO. Significant differences between the 10 mg-treated group and the control group are indicated (* P < 0.05, ** P < 0.02, *** P < 0.005). The 5 mg/kg and 20 mg/kg curves fall approximately halfway between the control and 10 mg/kg curves, whereas the 40 mg/kg curve is close�o the control. These curves are omitted for clarity.

addition to oxygen-derived free radicals. All of these

radicals have been shown to cause DNA strand breaks

and activate PARS (Zhang et aI., 1995; Szab6 et aI.,

1996). It has been recently demonstrated that during

stroke in mice, PARS is activated as revealed by a sub­

stantial increase of poly(ADP-ribose) polymer detected

by immunostaining (Elias son et aI., 1997).

A series of PARS inhibitors have been shown to pro-

200

ME .s 150 Ql E :::l '0 100 >

� .l!! c: 50

o Control 5mg/kg 10mg/kg 20mg/kg 40mg/kg

(n=14) (n=7) (n=7) (n=7) (n=7) FIG. 2. The effect of intraperitoneal administration of 3,4-dihydro 5-[4-(1-piperidinyl) butoxyj-1 (2H)-isoquinolinone on the infarct volume. The volumes of infarct are expressed as mean ± SO. Significant differences between the treated groups and the con­trol group are indicated (* P < 0.02, ** P < 0.002, *** P < 0.001).

J Cereb Blood Flow Metab. Vol. 17, No. 11. 1997

tect rat cortical cell cultures from NO-mediated neuro­

toxicity acting through NMDA-receptor with relative po­

tencies paralleling their ability as PARS inhibitors

(Zhang et aI., 1994). Several previous in vitro studies

have shown that PARS inhibitors are capable of protect­

ing cells against glutamate-mediated toxicity (Miller et

aI., 1993; Cosi et aI., 1994), NO-mediated toxicity (Ra­

dons et aI., 1994; Inada et aI., 1995), reactive oxygen

injury (Thies and Autor, 1991; Aalto and Raivio, 1993),

hydrogen peroxide-induced injury (Schraufstatter et aI.,

1986), and peroxynitrite-mediated injury (SzabO et aI.,

1996). These data show that PARS inhibition is neuro­

protective and indicate that NAD levels may be respon­

sible for this neuroprotection. It has been recently re­

ported that inhibitors of poly(ADP-ribosyl)ation also

provide good protection from NO-induced injury with

recovery of CA I-evoked responses in rat hippocampal

slices (Wallis et aI., 1993). In addition, pancreatic islet

cells from mice with an inactivated PARS gene do not

show NAD depletion after exposure to DNA-damaging

radicals, and are more resistant to the toxicity of both NO

and reactive oxygen intermediates (Heller et aI., 1995;

Wang et aI., 1995). The PARS inhibitors prevent the

rapid fall in NAD and ATP pools. This preservation of

the A TP pool apparently has a permissive effect on en­

ergy-dependent processes in the cell, which can salvage

injured tissue from intracellular energy depletion.

In our study, we treated animals with 3,4-dihydro 5-

[4-(1-piperidinyl) butoxy ]- 1 (2H)-isoquinolinone 2 hours

before the MCA occlusion and 2 hours after the MCA

occlusion (30 minutes after the reopen of the bilateral

CCA occlusion). There is evidence that single injections

of PARS inhibitors reduce the infarct size caused by

ischemia and reperfusion of the heart or skeletal muscle

in rabbits (Thiemermann et aI., 1997). In these studies, a

single injection of 3-aminobenzamide, another PARS in­

hibitor (10 mg/kg), either 1 minute before occlusion or 1

minute before reperfusion, causes similar reductions in

infarct size in the heart (32% or 42%). Other PARS

inhibitors, including 1,5-dihydroxyisoquinoline (1 mg/

kg), which belongs to a same series of PARS inhibitors

as 3,4 dihydro 5-4 [(l-piperidinyl) butoxy]- 1(2H)­

isoquinolinone, also reduce infarct size by 38% to 48%.

These findings suggest that PARS inhibitors are able to

salvage previously ischemic tissue, even when adminis­

tered with the onset of reperfusion .

The PARS inhibition has been shown to sensitize can­

cer cells for radiation and chemotherapy, as well as to

prevent damage to normal cells under ischemia­

reperfusion injury (Oleinick and Evans, 1985; Griffin et

aI., 1995; Thiemermann et aI., 1997). The exact mecha­

nism underlining these two paradoxical effects of PARS

inhibition is not clear. Accumulating evidence seems to

suggest that the effect of PARS inhibition depends on the

status of the cell in its replication cycle and the nature

NEUROPROTECTIVE EFFECTS OF INHIBITING POLY(ADP-RIBOSE) SYNTHETASE 1141

and extent of DNA damage that causes PARS activation.

It has been shown that 3-aminobenzamide has dramati­

cally different effects on x-ray-induced cytogenetic

damage in human lymphocytes, depending on the stage

of the cell cycle in which cells are irradiated (Wiencke

and Morgan, 1987). In rapidly dividing cells such as

cancer cells, PARS inhibition may delay the repair of

extensive DNA damage caused by radiation and may

lead to unchecked DNA replication causing cell death. In

quiescent cells, such as nondividing neurons, PARS in­

hibition may prevent energy depletion as a result of free­

radical damage to DNA that activates PARS, and thus

render cells more resistant to subsequent insults (Gaal et

aI., 1987). Secondly, PARS appears to be part of the

necessary repair mechanisms in cells irradiated with sub­

lethal to lethal doses. Excessive turnover of PARS,

which can deplete cells of their NAD pools, may be a

response to supralethal doses of ionizing radiation or

ischemic injury, and the loss of NAD may contribute to

cell death (Oleinick and Evans, 1985).

It is not clear why a high dose (40 mg/kg) of 3,4

dihydro 5-4[(l-piperidinyl) butoxy ]-1 (2H)-isoquino­

linone is less neuroprotective. The U-shaped dose­

response curve (Fig. 2) suggests dual effects of the com­

pound. In a study using mice whose PARS genes were

inactivated, it wasl'found that the infarct volume resulted

from MCA occlusion was substantially reduced (Elias­

son et aI., 1997). This suggests a secondary effect of this

compound at higher doses, which, instead of inhibiting

PARS, may exacerbate the neuronal damage.

Although this study demonstrates that the PARS in­

hibitors 3,4-dihydro 5-[4-( I-piperidinyl) butoxy ]- 1 (2H)­

isoquinolinone provides neuroprotection after cerebral

ischemia, the mechanism by which this compound pro­

vides this protection requires further investigation. Dem­

onstration that in vivo administration of PARS inhibitors

can attenuate the decrease of ATP/NAD levels during

ischemia, although complicated by the significant

changes in these substances that normally accompanies

hypoxia/ischemia, will provide additional support that

this compound can inhibit PARS in vivo. PARS plays an important role in the repair of DNA

damage. However, mice lacking PARS and poly(ADP­

ribosyl)ation are healthy and fertile. Embryonic fibro­

blasts derived from PARS knockout mice have been

shown to be able to repair DNA damage. These mice,

however, are susceptible to the spontaneous development

of skin disease, since -30% of older mice develop epi­

dermal hyperplasia (Wang et aI., 1995). Although lack of

PARS may lead to side effects, acute inhibition of PARS

can be of therapeutic value to prevent the ischemic tissue

injury. The substantial reduction in infarct volume ob­

served in this study indicates that PARS may play an

important role in ischemic celldamage.

REFERENCES

Aalto TK, Raivio KO (1993) Nucleotide depletion due to reactive oxygen metabolites in endothelial cells: effects of antioxidants and 3-aminobenzamide. Pediatr Res 34:572-576

Berger NA (1985) Poly(ADP-ribose) in the cellular response to DNA damage. Radiat Res 101:4-15

Bredt DS, Snyder SH (1989) Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci USA 86:9030-9033

Chen ST, Hsu CY, Hogan EL, et al (1986) A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction. Stroke 17:738-743

Cosi C, Suzuki H, Milani D, et al (1994) Poly(ADP-ribose) polymer­ase: early involvement in glutamate-induced neurotoxicity in cul­tured cerebellar granule cells. J Neurosci Res 39:38-46

Dawson VL, Dawson TM, Bartley DA, et al (1993) Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. J Neurosci 13:2651-2661

Dawson VL, Dawson TM, London ED, et al (1991) Nitric oxide me­diates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci USA 88:6368-6371

Eliasson MJL, Sampei K, Mandir AJ, et al (1997) Poly (ADP-ribose) polymerase gene disruption renders mice resistant to cerebral isch­emia. Nature Med (in press)

Gaal JC, Smith KR, Pearson CK (1987) Cellular euthanasia mediated by a nuclear enzyme: a central role for nuclear ADP-ribosylation in cellular metabolism. Trends BioI Sci 12:129-130

Garthwaite J, Charles SL, Chess-Williams R (1988) Endothelium­derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature 336: 385-388

Griffin RJ, Curtin NJ, Newell DR, et al (1995) The role of inhibitors poly(ADP-ribose) polymerase as resistance-modifying agents in cancer therapy. Biochimie 77:408-422

Heller B, Wang Z-Q, Wagner EF, et al (1995) Inactivation of the poly(ADP-ribose) polymerase gene affects oxygen radical and ni­tric oxide toxicity in islet cells. J BioI Chem 270: 11176-11180

Ignarro LJ, Buga GM, Wood KS, et al (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 84:9265-9269

Ikai K, Veda K, Hayaishi 0 (1980) Immunohistochemical demonstra­tion of poly(adenosine diphosphate-ribose) in nuclei of various rat tissues. J Histochem Cytochem 28:670-676

Inada C, Yamada K, Takane N, et al (1995) Poly(ADP-ribose) synthe­sis induced by nitric oxide in a mouse j3-cell line. Life Sci 56: 1467-1474

Liu TH, Beckman JS, Freeman BA, et al (1989) Polyethylene glycol­conjugated superoxide dismutase and catalase reduce ischemic brain injury. Am J Physio/ 256:H589-H593

Miller MS, Zobre C, Lewis M (1993) In vitro neuroprotective activity of inhibitors of poly-ADP-ribose polymerase. Soc Neurosci Abstr 19:1656

Ogura T, Takenouchi N, Yamaguchi M, et al (1990) Striking similarity of the distribution patterns of the poly(ADP-ribose) polymerase and DNA polymerase j3 among various mouse organs. Biochem Biophys Res Commun 172:377-384

Oleinick NL, Evans HH (1985) Poly(ADP-ribose) and the response of cells to ionizing radiation. Radiat Res 101:29-46

Palmer RMJ, Ferrige AG, Moncada S (1987) Nitric oxide release ac­counts for the biological activity of endothelium-derived relaxing factor. Nature 327:524-526

Radons J, Heller B, Burkle A, et al (1994) Nitric oxide toxicity in islet cells involves poly(ADP-ribose) polymerase activation and con­comitant NAD+ depletion. Biochem Biophys Res Commun 199: 1270-1277

Satoh MS, Lindahl T (1992) Role of poly(ADP-ribose) formation in DNA repair. Nature 356:356-358

Schraufstatter IU, Hyslop PA, Hinshaw DB, et al (1986) Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose) polymerase. Proc Natl Acad Sci USA 83:4908-4912

Suto MJ, Turner WR, Werbel LM (1993) Substituted dihydroisoquino-

J Cereb Blood Flow Metab. Vol. 17, No. II, 1997

1142 K. TAKAHASH1 ET AL.

linones and related compounds as potentiators of the lethal effects of radiation and certain chemotherapeutic agents: selected com­pounds, analogs and process. United States Patent 5,177,075

Swanson RA, Morton MT, Tsao-Wu G, et al (1990) A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10:290-293

Szabo C, Zingarelli B, O'Connor M, et al (1996) DNA strand breakage, activation of poly(ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite. Proc Nat! Acad Sci

USA 93:1753-1758 Thiemermann C, Bowes J, Myint FP, et al (1997) Inhibition of the

activity of poly(ADP ribose) synthetase reduces ischemia-reper­fusion injury in the heart and skeletal muscle. Proc Natl Acad Sci USA 94:679-683

Thies RL, Autor AP (1991) Reactive oxygen injury to cultured pulmo­nary artery endothelial cells: mediation by poly(ADP-ribose) poly­merase activation causing NAD depletion and altered energy bal­ance. Arch Biochem Biophys 286:353-363

J Cereb Blood Flow Metab, Vol. 17, No. II, 1997

Wallis RA, Panizzon, KL, Henry D, et al (1993) Neuroprotection against nitric oxide injury with inhibitors of ADP-ribosylation. Neuroreport 5:245-248

Wang Z-Q, Auer B, Stingl L, et al (1995) Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease. Genes Devel 9:509-520

Wiencke JK, Morgan WF (1987) Cell cycle-dependent potentiation of x-ray-induced chromosomal aberrations by 3-amonobenzamide. Biochem Biophys Res Commun 143:372-376

Yip PK, He YY, Hsu CY, et al (1991) Effect of plasma glucose on infarct size in focal cerebral ischemia-reperfusion. Neurology 41: 899-905

Zhang J, Dawson VL, Dawson TM, et al (1994) Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 263 :687-689

Zhang J, Pieper A, Snyder SH (1995) Poly(ADP-ribose) synthetase activation: an early indicator of neurotoxic DNA damage. J Neu­rochem 65:1411-1414