Antioxidant nutrients and hypoxia/ischemia brain injury in rodents

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  • 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 Womens 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*/thatis, 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 associate