Antioxidant nutrients and hypoxia/ischemia brain injury inrodents

Katsumi Ikeda a,c,*, Hiroko Negishi b,c, Yukio Yamori c

a School of Human Environmental Sciences, Mukogawa Women’s University, Ikebiraki-cho, Nishinomiya, Japanb Graduate School of Human Environmental Studies, Kyoto University, Kyoto, Japan

c WHO Collaborating Center for Research on Primary Prevention of Cardiovascular Diseases, Kyoto, Japan

Abstract

Cerebral ischemia and recirculation cause delayed neuronal death in rodents, such as Mongolian gerbils and stroke-

prone spontaneously hypertensive rats (SHRSP), which were used as an experimental stroke model. It was documented

that an enhanced nitric oxide production, the occurrence of apoptosis, and an attenuated redox regulatory system

contribute to the development of delayed neuronal death. Many studies have suggested the beneficial antioxidant

effects of antioxidant nutrients such as vitamin E, green tea extract, ginkgo biloba extract, resveratrol and niacin in

cerebral ischemia and recirculation brain injury. These results are important in light of an attenuation of the deleterious

consequences of oxidative stress in ischemia and recirculation injury.

# 2003 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Mongolian gerbil; SHRSP; Delayed neuronal death; Antioxidant nutrients

1. Introduction

A common cause of stroke, cerebral infarct, is

atherosclerosis that forms arterial thrombosis. An

arterial thrombosis may transiently or perma-

nently block the artery, and often leads to ischemic

damage of the tissue supplied by the artery*/that

is, an infarct. Cerebral infarct is the third leading

cause of death in most developed countries, and

the leading cause of disability in adults.

Several studies have suggested a relation be-

tween cerebral ischemia and oxidative stress in

humans (Chang et al., 1998; Hume et al., 1982;

Spranger et al., 1997). Antioxidants have been

evaluated as one of the neuroprotective agents in

stroke (Cherubini et al., 2000). In experimental

studies, to assess the neuroprotective agents, such

as antioxidants, we were able to use a transient

ischemia model in rodents. Recently, it appears

that antioxidant nutrients, especially those from

food sources, have important roles in preventing* Corresponding author. Tel.: �/81-798-45-9956.

E-mail address: ikeda@mwu.mukogawa-u.ac.jp (K. Ikeda).

Toxicology 189 (2003) 55�/61

www.elsevier.com/locate/toxicol

0300-483X/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.

doi:10.1016/S0300-483X(03)00152-5

pathogenic processes related to ischemia/reperfu-sion injury. In the present article, we summarized

the preventive effects of antioxidant nutrients on

the occurrence of neuronal cell death in rodent

models.

2. Hypoxia/ischemia brain injury in Mongolian

gerbils and SHRSP

It is well recognized that transient ischemia

induced by bilateral common carotid artery occlu-

sion followed by oxygen reperfusion induces

neuronal death in Mongolian gerbils as well as in

genetic hypertensive rats. When Mongolian gerbils

were subjected to bilateral carotid occlusion for 5

min, the death of the CA1 pyramidal cells becameapparent 2 days following ischemia. This change in

CA1 was called delayed neuronal death (Kirino,

1982). Several studies have reported evidence of

increased oxygen free radicals during ischemia/

reperfusion of the gerbil brain (Cao et al., 1988;

Delbarre et al., 1991; Hall et al., 1993).

On the other hand, spontaneously hypertensive

rats (SHR) characterized by spontaneous hyper-tension with age have been used as a model of

human essential hypertension. In 1973, stroke-

prone SHR (SHRSP) were selected from SHR

substrains. SHRSP show severe hypertension and

the development of stroke, and are regarded as a

stroke model. Many studies documented the

occurrence of delayed neuronal death in SHRSP.

Gemba et al. (1992) showed that cerebral ischemiafor 20 min in SHRSP induces massive efflux of

glutamate, causing delayed neuronal death.

SHRSP neurons are more sensitive than Wistar

Kyoto rat (WKY) neurons to hypoxia and oxygen

reperfusion (Tagami et al., 1999a). The glial

endothelin/nitric oxide system participates in hip-

pocampus CA1 neuronal death of SHRSP follow-

ing transient forebrain ischemia (Yamashita et al.,1995). We observed that oxygen radical generation

occurs after reperfusion (Negishi et al., 2001;

Tagami et al., 1997). These reports suggested

that reactive oxygen species (ROS) play an im-

portant role in the occurrence of delayed neuronal

death in SHRSP.

3. Contributing factors of neuronal vulnerability toischemia injury in SHRSP and Mongolian gerbils

Several mechanisms participate in neuronal

vulnerability in SHRSP subjected to ischemia

and recirculation. In this article, we introduce the

contribution of thioredoxins (TRX), Bcl-2 and

nitric oxide on the development of neuronal death

in SHRSP subjected to transient ischemia.

3.1. TRX

TRX are 12-kDa redox regulatory proteins

known to be present in all eukaryotic and prokar-

yotic organisms (Gleason and Holmgren, 1988).

Several studies have suggested that TRX induction

is accompanied by reactive oxygen intermediate(ROI) overproduction and may play an important

role not only in scavenging ROI, but also in signal

transduction during ischemia (Takagi et al., 1998,

1999). Previous studies documented that redox

regulatory function metabolism, such as the ex-

pression of thioredoxin, in SHRSP cultured cor-

tical neurons was markedly reduced by oxygen

stimulation after hypoxia (Yamagata et al., 2000).TRX mRNA expression in SHRSP was also

significantly lower than in normotensive WKY.

3.2. Bcl-2

Apoptosis is known as programmed cell death.

The bcl-2 gene is antiapoptopic in mammalian

cells. Recent studies have shown that cytochrome c

release from the mitochondria is a key componentin the activation of caspases, leading to apoptosis.

Bcl-2 localizes predominantly to the outer mito-

chonrial membrane, but also to the nuclear and

endoplasmic reticulum membranes. Bcl-2 acts on

mitochondria to prevent the release of cytochrome

c and inhibits caspase-3 activation. Caspases are

cysteine proteases that cleave after aspartic acid

residues. A member of this family, caspase-3, hasbeen identified as being a key mediator of apop-

tosis in mammalian cells.

We observed the expression of bcl-2 mRNA in

SHRSP after ischemia and reperfusion. The re-

duction of bcl-2 mRNA expression in SHRSP was

significantly greater than in WKY. Western blot

K. Ikeda et al. / Toxicology 189 (2003) 55�/6156

analysis shows that bcl-2 at the protein level inSHRSP was also decreased at 24 h after reperfu-

sion (data not shown). The expression of bcl-2

mRNA induced by reoxygenation in SHRSP was

significantly lower than that detected in WKY,

suggesting apoptosis more readily occurs in

SHRSP neurons.

3.3. Nitric oxide (NO)

NO induces apoptosis of a variety of types of

cultured cells including neurons and may contri-

bute to the neurons in several disorders including

ischemic stroke. Oxidative damage to cellular

proteins and nucleic acids can trigger an apoptotic

cascade involving the release of cytochrome c and

activation of caspases. A previous study demon-

strated that cerebral ischemia followed by oxygenreperfusion induced apoptosis in hippocampal

neurons in SHRSP (Tagami et al., 1999b). The

present findings showed that the expressions of

nNOS and iNOS mRNA in SHRSP were signifi-

cantly increased at 12 h after reperfusion.

From these results, it is suggested that the

contributing factors of neuronal vulnerability in

ischemic injury in SHRSP are as follows: (1)Hippocampal neuronal damage following ische-

mia and recirculation in SHRSP is partially caused

by the increase in nitric oxide and hydroxyl

radicals during ischemia and recirculation. (2)

Reduced bcl-2 mRNA expression following ische-

mia and reperfusion suggests that anti-apoptopic

action is more attenuated in the hippocampus of

SHRSP than in WKY.In Mongolian gerbils, Antonawich et al. (1998)

documented that bcl-2-associated X protein Bax

levels were markedly increased at 6 h after

transient ischemia. Hayashi et al. (2001) suggested

that the inhibition of caspase-1 activity amelio-

rates the ischemic injury by inhibiting the activity

of IL-1beta. These findings suggest that apoptosis

contributes to the occurrence of delayed neuronaldeath in Mongolian gerbils. Furthermore, NG-

nitro-L-arginine, a nitric oxide synthase inhibitor,

reduced the occurrence of neuronal death in the

lateral CA1 subfield of Mongolian gerbils sub-

jected to 4 min of transient ischemia, suggesting an

important role of nitric oxide in the development

of neuronal injury after global ischemia (Naka-gomi et al., 1997). Increases in nitrite and nitrate

were observed after cerebral ischemia in the

hippocampus of Mongolian gerbils (Calapai et

al., 2000). In neuronal NOS null mice, a deficiency

in neuronal NO production slowed the develop-

ment of apoptotic cell death after ischemic injury

and was associated with preserved bcl-2 levels and

delayed activation of effector caspases (Elibol etal., 2001).

This attenuated redox regulatory system, which

enhanced nitric oxide production and the occur-

rence of apoptosis, suggests that increased oxida-

tive stress may participate in the development of

neuronal death in the hippocampal CA1 in

SHRSP and Mongolian gerbils subjected to ische-

mia and reperfusion.

4. Antioxidant nutrients

Oxidative stress is a function of balance between

pro-oxidants, such as ROS and antioxidants

scavenging them. Neuronal damage following

transient cerebral ischemia is mediated by variousmechanisms, among which oxygen radical-

mediated processes play a central role. Ischemia

and subsequent recirculation provide circum-

stances that favor their production. Therefore,

antioxidants have been evaluated as neuroprotec-

tive agents and are able to reduce the cerebral

damage in ischemia and reperfusion. It is sug-

gested that antioxidant defense against toxic oxy-gen intermediates is heavily influenced by nutrition

in humans (Elsayed, 2001). In the case of rodent

models, protection against ischemia-reperfusion

induced oxidative stress by antioxidant nutrients,

such as vitamin E, green tea extract, Ginkgo

biloba extract and red wine/resveratrol, has been

documented.

4.1. Vitamin E

Vitamin E cannot be produced in the body of

animals and humans. Foodstuffs, such as vegeta-

ble oils, nuts and cod roe, are good sources of

Vitamin E. Vitamin E is a fat-soluble vitamin that

exists in eight different forms. Alpha-tocopherol is

K. Ikeda et al. / Toxicology 189 (2003) 55�/61 57

the most active form of vitamin E in humans, andis a powerful antioxidant protecting unsaturated

fatty acids, protein and DNA from oxidation.

It has been reported that the majority of

antioxidants are reduced immediately after an

acute ischemic stroke (Cherubini et al., 2000).

Chang et al. (1998) observed that vitamin E in

the plasma of ischemic-stroke patients was sig-

nificantly lower than that of the controls. Severalstudies documented the preventive effects of vita-

min E in neuronal cell death caused by ischemia

and reperfusion in rodent models. Tagami et al.

(1998) studied hypoxia and oxygen reperfusion

using cortical neurons isolated from SHRSP. They

reported that antioxidants, including vitamin E,

reacted with the radicals, thereby preventing

apoptosis and necrosis in vitro. It was furtherobserved that vitamin E reacts with the radicals

and prevents neuronal apoptosis caused by cere-

bral ischemia and reperfusion in SHRSP (Tagami

et al., 1999a).

4.2. Green tea extract

Green tea contains antioxidant polyphenols,such as catechins and flavonols. It has been

suggested that tea polyphenols scavenge ROS

(Yoshino and Murakami, 1998; Scott et al.,

1993). Recently, Hong et al. showed that green

tea extract prevented cerebral ischemia damage

caused by global ischemia and recirculation in

Mongolian gerbils and rats (Hong et al., 2000a,b).

(�/)-Epigallocatechin gallate (EGCG) has a potentantioxidant property in a green tea polyphone. Lee

et al. (2000) observed that EGCG had a neuro-

protective effect against neuronal damage follow-

ing global ischemia in Mongolian gerbils.

4.3. Ginkgo biloba extract

Ginkgo biloba is extracted from the leaves and

nuts of the Ginkgo biloba tree. Ginkgo bilobaextracts contain flavone glycosides (primarily

composed of quercetin, kaempferol, rutin and

myricetin) and terpene lactones, which decrease

free radical release (Pietri et al., 1997). A free

radical scavenging action of Ginkgo biloba extract

was reported by Louajri et al. (2001). Calipee et al.

showed that delayed neuronal death in the CA1 ofthe hippocampus was attenuated by the highest

dose of this extract in Mongolian gerbils (Calapai

et al., 2000). Ginkgo biloba extract also showed

reductions in stroke infarct volume in mice sub-

jected to 45 min of middle cerebral artery occlu-

sion and reperfusion (Clark et al., 2001).

4.4. Red wine/resveratrol

Recent studies have suggested that regular

consumption of red wine reduces the risk of

atherosclerosis and coronary heart diseases. It is

considered that this effect is attributed in part to

the antioxidant properties of polyphenolic com-

pounds such as resveratrol (3,5,4?-trihydroxy-

trans-stilbene), one of the major antioxidant con-stituents found in the skin of grapes. Resveratrol

has been considered responsible, in part, for the

protective effects of red wine consumption against

coronary heart disease. Our recent study docu-

mented that resveratrol was a powerful antioxi-

dant, able to interfere with advanced glycation end

products, mediated oxidative DNA damage, and

was a useful agent against vascular diseases whereROS were involved in hypertension (Mizutani et

al., 2000a,b). Few studies of the effects of resver-

atrol in cerebral ischemia and reperfusion injury

have been made. Huang’s study suggests that

resveratrol is a potent neuroprotective agent in

focal cerebral ischemia caused by middle cerebral

artery occlusion for 1 and 24 h of recirculation in

Long�/Evans rats (Huang et al., 2001).

4.5. Niacin (Vitamin B3)

Niacin (Vitamin B3) is the common name for

two compounds: nicotinic acid and nicotinamide.

Niacin is a water-soluble vitamin that participates

in many metabolic functions. Recent evidence

suggests a neuroprotective effect of niacin in rats.

Delayed treatment with nicotinamide protects ratssubjected to permanent middle cerebral artery

occlusion (Sakakibara et al., 2000). It was also

reported that nicotinamide is a robust neuropro-

tective agent against ischemia/reperfusion-induced

brain injury in rats (Mokudai et al., 2000; May-

nard et al., 2001). Nicotinamide showed significant

K. Ikeda et al. / Toxicology 189 (2003) 55�/6158

inhibition of oxidative damage induced by ROSgenerated by ascorbate�/Fe2� and photosensitiza-

tion systems in rat brain mitochondria (Kamat

and Devasagayam, 1999). However, the mechan-

ism of action underlying the neuroprotection

observed with niacin remains to be clarified.

4.6. Isoflavones

It is known that isoflavones in plants show an

estrogenic action (Kurzer and Xu, 1997). Soybeans

and soy foods are the most significant dietary

sources of isoflavones (Messina and Messina,

2000) and contain three main isoflavones, daid-

zein, genistein and glycitein. They are present as

acetylglucosides and malonylglucosides (Liggins et

al., 1998).Many studies have shown isoflavones to exert

antioxidant effects. Genistein potently scavenged

both hydrogen peroxide in the medium and super-

oxide anions generated by xanthine/xanthine ox-

idase (IC50�/1�/2.5 mM), and daidzein showed a

moderate inhibitory effect (IC50�/5 mM) (Wei et

al., 1996). In humans, Jenkins et al. (2000)

observed that high isoflavone intake might de-crease the risk of cardiovascular disease by redu-

cing oxidized LDL. Djuric et al. reported that soy

isoflavone supplementation decreased the levels of

oxidative DNA damage (Djuric et al., 2001).

However, as the preventive effects of isoflavones

on ischemic/reperfusion neuronal cell injury in

rodent models are not well understood, further

studies are needed.

5. Conclusion

Cerebral ischemic injury caused by transient

ischemia shows delayed neuronal death in rodent

models. The occurrence of delayed neuronal death

is the results of several factors, an enhanced nitric

oxide production and the attenutated redox reg-ulatory system. Enhanced nitric oxide production

is involved in the development of apoptosis. The

attenuated redox regulatory system leads to in-

creasing oxidative stress. Therefore, antioxidant

agents, such as antioxidant nutrients, prevent

brain injury caused by ischemia and recirculation.

Vitamin E, Ginkgo biloba extract, resveratrol andvitamin B3 were shown to have preventive effects

in delayed neuronal death caused by transient

ischemia in rodent models.

Stroke is the leading cause of disability. The

societal cost attributable to stroke is immense and

encourages the medical community to seek new

therapies that can reduce stroke frequency and

impact. It is, therefore, an ideal target for effectiveprevention strategies that are simple and inexpen-

sive (Chaturvedi et al., 1999). Stroke is multi-

factorial in its etiology, but there remains some

potential for disease prevention by dietary manip-

ulation. For stroke, dietary factors play important

roles in modulating blood lipids and their propen-

sity for oxidation (Mera, 1994). The antioxidant

nutrients presented in this study may have thepotential for stroke prevention.

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