Antioxidant nutrients and hypoxia/ischemia brain injury in rodents

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<ul><li><p>Antioxidant nutrients and hypoxia/ischemia brain injury inrodents</p><p>Katsumi Ikeda a,c,*, Hiroko Negishi b,c, Yukio Yamori c</p><p>a School of Human Environmental Sciences, Mukogawa Womens University, Ikebiraki-cho, Nishinomiya, Japanb Graduate School of Human Environmental Studies, Kyoto University, Kyoto, Japan</p><p>c WHO Collaborating Center for Research on Primary Prevention of Cardiovascular Diseases, Kyoto, Japan</p><p>Abstract</p><p>Cerebral ischemia and recirculation cause delayed neuronal death in rodents, such as Mongolian gerbils and stroke-</p><p>prone spontaneously hypertensive rats (SHRSP), which were used as an experimental stroke model. It was documented</p><p>that an enhanced nitric oxide production, the occurrence of apoptosis, and an attenuated redox regulatory system</p><p>contribute to the development of delayed neuronal death. Many studies have suggested the beneficial antioxidant</p><p>effects of antioxidant nutrients such as vitamin E, green tea extract, ginkgo biloba extract, resveratrol and niacin in</p><p>cerebral ischemia and recirculation brain injury. These results are important in light of an attenuation of the deleterious</p><p>consequences of oxidative stress in ischemia and recirculation injury.</p><p># 2003 Elsevier Science Ireland Ltd. All rights reserved.</p><p>Keywords: Mongolian gerbil; SHRSP; Delayed neuronal death; Antioxidant nutrients</p><p>1. Introduction</p><p>A common cause of stroke, cerebral infarct, is</p><p>atherosclerosis that forms arterial thrombosis. An</p><p>arterial thrombosis may transiently or perma-</p><p>nently block the artery, and often leads to ischemic</p><p>damage of the tissue supplied by the artery*/thatis, an infarct. Cerebral infarct is the third leading</p><p>cause of death in most developed countries, and</p><p>the leading cause of disability in adults.</p><p>Several studies have suggested a relation be-</p><p>tween cerebral ischemia and oxidative stress in</p><p>humans (Chang et al., 1998; Hume et al., 1982;</p><p>Spranger et al., 1997). Antioxidants have been</p><p>evaluated as one of the neuroprotective agents in</p><p>stroke (Cherubini et al., 2000). In experimental</p><p>studies, to assess the neuroprotective agents, such</p><p>as antioxidants, we were able to use a transient</p><p>ischemia model in rodents. Recently, it appears</p><p>that antioxidant nutrients, especially those from</p><p>food sources, have important roles in preventing* Corresponding author. Tel.: /81-798-45-9956.E-mail address: (K. Ikeda).</p><p>Toxicology 189 (2003) 55/61</p><p></p><p>0300-483X/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.doi:10.1016/S0300-483X(03)00152-5</p></li><li><p>pathogenic processes related to ischemia/reperfu-sion injury. In the present article, we summarized</p><p>the preventive effects of antioxidant nutrients on</p><p>the occurrence of neuronal cell death in rodent</p><p>models.</p><p>2. Hypoxia/ischemia brain injury in Mongolian</p><p>gerbils and SHRSP</p><p>It is well recognized that transient ischemia</p><p>induced by bilateral common carotid artery occlu-</p><p>sion followed by oxygen reperfusion induces</p><p>neuronal death in Mongolian gerbils as well as in</p><p>genetic hypertensive rats. When Mongolian gerbils</p><p>were subjected to bilateral carotid occlusion for 5</p><p>min, the death of the CA1 pyramidal cells becameapparent 2 days following ischemia. This change in</p><p>CA1 was called delayed neuronal death (Kirino,</p><p>1982). Several studies have reported evidence of</p><p>increased oxygen free radicals during ischemia/</p><p>reperfusion of the gerbil brain (Cao et al., 1988;</p><p>Delbarre et al., 1991; Hall et al., 1993).</p><p>On the other hand, spontaneously hypertensive</p><p>rats (SHR) characterized by spontaneous hyper-tension with age have been used as a model of</p><p>human essential hypertension. In 1973, stroke-</p><p>prone SHR (SHRSP) were selected from SHR</p><p>substrains. SHRSP show severe hypertension and</p><p>the development of stroke, and are regarded as a</p><p>stroke model. Many studies documented the</p><p>occurrence of delayed neuronal death in SHRSP.</p><p>Gemba et al. (1992) showed that cerebral ischemiafor 20 min in SHRSP induces massive efflux of</p><p>glutamate, causing delayed neuronal death.</p><p>SHRSP neurons are more sensitive than Wistar</p><p>Kyoto rat (WKY) neurons to hypoxia and oxygen</p><p>reperfusion (Tagami et al., 1999a). The glial</p><p>endothelin/nitric oxide system participates in hip-</p><p>pocampus CA1 neuronal death of SHRSP follow-</p><p>ing transient forebrain ischemia (Yamashita et al.,1995). We observed that oxygen radical generation</p><p>occurs after reperfusion (Negishi et al., 2001;</p><p>Tagami et al., 1997). These reports suggested</p><p>that reactive oxygen species (ROS) play an im-</p><p>portant role in the occurrence of delayed neuronal</p><p>death in SHRSP.</p><p>3. Contributing factors of neuronal vulnerability toischemia injury in SHRSP and Mongolian gerbils</p><p>Several mechanisms participate in neuronal</p><p>vulnerability in SHRSP subjected to ischemia</p><p>and recirculation. In this article, we introduce the</p><p>contribution of thioredoxins (TRX), Bcl-2 and</p><p>nitric oxide on the development of neuronal death</p><p>in SHRSP subjected to transient ischemia.</p><p>3.1. TRX</p><p>TRX are 12-kDa redox regulatory proteins</p><p>known to be present in all eukaryotic and prokar-</p><p>yotic organisms (Gleason and Holmgren, 1988).</p><p>Several studies have suggested that TRX induction</p><p>is accompanied by reactive oxygen intermediate(ROI) overproduction and may play an important</p><p>role not only in scavenging ROI, but also in signal</p><p>transduction during ischemia (Takagi et al., 1998,</p><p>1999). Previous studies documented that redox</p><p>regulatory function metabolism, such as the ex-</p><p>pression of thioredoxin, in SHRSP cultured cor-</p><p>tical neurons was markedly reduced by oxygen</p><p>stimulation after hypoxia (Yamagata et al., 2000).TRX mRNA expression in SHRSP was also</p><p>significantly lower than in normotensive WKY.</p><p>3.2. Bcl-2</p><p>Apoptosis is known as programmed cell death.</p><p>The bcl-2 gene is antiapoptopic in mammalian</p><p>cells. Recent studies have shown that cytochrome c</p><p>release from the mitochondria is a key componentin the activation of caspases, leading to apoptosis.</p><p>Bcl-2 localizes predominantly to the outer mito-</p><p>chonrial membrane, but also to the nuclear and</p><p>endoplasmic reticulum membranes. Bcl-2 acts on</p><p>mitochondria to prevent the release of cytochrome</p><p>c and inhibits caspase-3 activation. Caspases are</p><p>cysteine proteases that cleave after aspartic acid</p><p>residues. A member of this family, caspase-3, hasbeen identified as being a key mediator of apop-</p><p>tosis in mammalian cells.</p><p>We observed the expression of bcl-2 mRNA in</p><p>SHRSP after ischemia and reperfusion. The re-</p><p>duction of bcl-2 mRNA expression in SHRSP was</p><p>significantly greater than in WKY. Western blot</p><p>K. Ikeda et al. / Toxicology 189 (2003) 55/6156</p></li><li><p>analysis shows that bcl-2 at the protein level inSHRSP was also decreased at 24 h after reperfu-</p><p>sion (data not shown). The expression of bcl-2</p><p>mRNA induced by reoxygenation in SHRSP was</p><p>significantly lower than that detected in WKY,</p><p>suggesting apoptosis more readily occurs in</p><p>SHRSP neurons.</p><p>3.3. Nitric oxide (NO)</p><p>NO induces apoptosis of a variety of types of</p><p>cultured cells including neurons and may contri-</p><p>bute to the neurons in several disorders including</p><p>ischemic stroke. Oxidative damage to cellular</p><p>proteins and nucleic acids can trigger an apoptotic</p><p>cascade involving the release of cytochrome c and</p><p>activation of caspases. A previous study demon-</p><p>strated that cerebral ischemia followed by oxygenreperfusion induced apoptosis in hippocampal</p><p>neurons in SHRSP (Tagami et al., 1999b). The</p><p>present findings showed that the expressions of</p><p>nNOS and iNOS mRNA in SHRSP were signifi-</p><p>cantly increased at 12 h after reperfusion.</p><p>From these results, it is suggested that the</p><p>contributing factors of neuronal vulnerability in</p><p>ischemic injury in SHRSP are as follows: (1)Hippocampal neuronal damage following ische-</p><p>mia and recirculation in SHRSP is partially caused</p><p>by the increase in nitric oxide and hydroxyl</p><p>radicals during ischemia and recirculation. (2)</p><p>Reduced bcl-2 mRNA expression following ische-</p><p>mia and reperfusion suggests that anti-apoptopic</p><p>action is more attenuated in the hippocampus of</p><p>SHRSP than in WKY.In Mongolian gerbils, Antonawich et al. (1998)</p><p>documented that bcl-2-associated X protein Bax</p><p>levels were markedly increased at 6 h after</p><p>transient ischemia. Hayashi et al. (2001) suggested</p><p>that the inhibition of caspase-1 activity amelio-</p><p>rates the ischemic injury by inhibiting the activity</p><p>of IL-1beta. These findings suggest that apoptosis</p><p>contributes to the occurrence of delayed neuronaldeath in Mongolian gerbils. Furthermore, NG-</p><p>nitro-L-arginine, a nitric oxide synthase inhibitor,</p><p>reduced the occurrence of neuronal death in the</p><p>lateral CA1 subfield of Mongolian gerbils sub-</p><p>jected to 4 min of transient ischemia, suggesting an</p><p>important role of nitric oxide in the development</p><p>of neuronal injury after global ischemia (Naka-gomi et al., 1997). Increases in nitrite and nitrate</p><p>were observed after cerebral ischemia in the</p><p>hippocampus of Mongolian gerbils (Calapai et</p><p>al., 2000). In neuronal NOS null mice, a deficiency</p><p>in neuronal NO production slowed the develop-</p><p>ment of apoptotic cell death after ischemic injury</p><p>and was associated with preserved bcl-2 levels and</p><p>delayed activation of effector caspases (Elibol etal., 2001).</p><p>This attenuated redox regulatory system, which</p><p>enhanced nitric oxide production and the occur-</p><p>rence of apoptosis, suggests that increased oxida-</p><p>tive stress may participate in the development of</p><p>neuronal death in the hippocampal CA1 in</p><p>SHRSP and Mongolian gerbils subjected to ische-</p><p>mia and reperfusion.</p><p>4. Antioxidant nutrients</p><p>Oxidative stress is a function of balance between</p><p>pro-oxidants, such as ROS and antioxidants</p><p>scavenging them. Neuronal damage following</p><p>transient cerebral ischemia is mediated by variousmechanisms, among which oxygen radical-</p><p>mediated processes play a central role. Ischemia</p><p>and subsequent recirculation provide circum-</p><p>stances that favor their production. Therefore,</p><p>antioxidants have been evaluated as neuroprotec-</p><p>tive agents and are able to reduce the cerebral</p><p>damage in ischemia and reperfusion. It is sug-</p><p>gested that antioxidant defense against toxic oxy-gen intermediates is heavily influenced by nutrition</p><p>in humans (Elsayed, 2001). In the case of rodent</p><p>models, protection against ischemia-reperfusion</p><p>induced oxidative stress by antioxidant nutrients,</p><p>such as vitamin E, green tea extract, Ginkgo</p><p>biloba extract and red wine/resveratrol, has been</p><p>documented.</p><p>4.1. Vitamin E</p><p>Vitamin E cannot be produced in the body of</p><p>animals and humans. Foodstuffs, such as vegeta-</p><p>ble oils, nuts and cod roe, are good sources of</p><p>Vitamin E. Vitamin E is a fat-soluble vitamin that</p><p>exists in eight different forms. Alpha-tocopherol is</p><p>K. Ikeda et al. / Toxicology 189 (2003) 55/61 57</p></li><li><p>the most active form of vitamin E in humans, andis a powerful antioxidant protecting unsaturated</p><p>fatty acids, protein and DNA from oxidation.</p><p>It has been reported that the majority of</p><p>antioxidants are reduced immediately after an</p><p>acute ischemic stroke (Cherubini et al., 2000).</p><p>Chang et al. (1998) observed that vitamin E in</p><p>the plasma of ischemic-stroke patients was sig-</p><p>nificantly lower than that of the controls. Severalstudies documented the preventive effects of vita-</p><p>min E in neuronal cell death caused by ischemia</p><p>and reperfusion in rodent models. Tagami et al.</p><p>(1998) studied hypoxia and oxygen reperfusion</p><p>using cortical neurons isolated from SHRSP. They</p><p>reported that antioxidants, including vitamin E,</p><p>reacted with the radicals, thereby preventing</p><p>apoptosis and necrosis in vitro. It was furtherobserved that vitamin E reacts with the radicals</p><p>and prevents neuronal apoptosis caused by cere-</p><p>bral ischemia and reperfusion in SHRSP (Tagami</p><p>et al., 1999a).</p><p>4.2. Green tea extract</p><p>Green tea contains antioxidant polyphenols,such as catechins and flavonols. It has been</p><p>suggested that tea polyphenols scavenge ROS</p><p>(Yoshino and Murakami, 1998; Scott et al.,</p><p>1993). Recently, Hong et al. showed that green</p><p>tea extract prevented cerebral ischemia damage</p><p>caused by global ischemia and recirculation in</p><p>Mongolian gerbils and rats (Hong et al., 2000a,b).</p><p>(/)-Epigallocatechin gallate (EGCG) has a potentantioxidant property in a green tea polyphone. Lee</p><p>et al. (2000) observed that EGCG had a neuro-</p><p>protective effect against neuronal damage follow-</p><p>ing global ischemia in Mongolian gerbils.</p><p>4.3. Ginkgo biloba extract</p><p>Ginkgo biloba is extracted from the leaves and</p><p>nuts of the Ginkgo biloba tree. Ginkgo bilobaextracts contain flavone glycosides (primarily</p><p>composed of quercetin, kaempferol, rutin and</p><p>myricetin) and terpene lactones, which decrease</p><p>free radical release (Pietri et al., 1997). A free</p><p>radical scavenging action of Ginkgo biloba extract</p><p>was reported by Louajri et al. (2001). Calipee et al.</p><p>showed that delayed neuronal death in the CA1 ofthe hippocampus was attenuated by the highest</p><p>dose of this extract in Mongolian gerbils (Calapai</p><p>et al., 2000). Ginkgo biloba extract also showed</p><p>reductions in stroke infarct volume in mice sub-</p><p>jected to 45 min of middle cerebral artery occlu-</p><p>sion and reperfusion (Clark et al., 2001).</p><p>4.4. Red wine/resveratrol</p><p>Recent studies have suggested that regular</p><p>consumption of red wine reduces the risk of</p><p>atherosclerosis and coronary heart diseases. It is</p><p>considered that this effect is attributed in part to</p><p>the antioxidant properties of polyphenolic com-</p><p>pounds such as resveratrol (3,5,4?-trihydroxy-trans-stilbene), one of the major antioxidant con-stituents found in the skin of grapes. Resveratrol</p><p>has been considered responsible, in part, for the</p><p>protective effects of red wine consumption against</p><p>coronary heart disease. Our recent study docu-</p><p>mented that resveratrol was a powerful antioxi-</p><p>dant, able to interfere with advanced glycation end</p><p>products, mediated oxidative DNA damage, and</p><p>was a useful agent against vascular diseases whereROS were involved in hypertension (Mizutani et</p><p>al., 2000a,b). Few studies of the effects of resver-</p><p>atrol in cerebral ischemia and reperfusion injury</p><p>have been made. Huangs study suggests that</p><p>resveratrol is a potent neuroprotective agent in</p><p>focal cerebral ischemia caused by middle cerebral</p><p>artery occlusion for 1 and 24 h of recirculation in</p><p>Long/Evans rats (Huang et al., 2001).</p><p>4.5. Niacin (Vitamin B3)</p><p>Niacin (Vitamin B3) is the common name for</p><p>two compounds: nicotinic acid and nicotinamide.</p><p>Niacin is a water-soluble vitamin that participates</p><p>in many metabolic functions. Recent evidence</p><p>suggests a neuroprotective effect of niacin in rats.</p><p>Delayed treatment with nicotinamide protects ratssubjected to permanent middle cerebral artery</p><p>occlusion (Sakakibara et al., 2000). It was also</p><p>reported that nicotinamide is a robust neuropro-</p><p>tective agent against ischemia/reperfusion-induced</p><p>brain injury in rats (Mokudai et al., 2000; May-</p><p>nard et al., 2001). Nicotinamide showed significant</p><p>K. Ikeda et al. / Toxicology 189 (2003) 55/6158</p></li><li><p>inhibition of oxidative damage induced by ROSgenerated by ascorbate/Fe2 and photosensitiza-tion systems in rat brain mitochondria (Kamat</p><p>and Devasagayam, 1999). However, the mechan-</p><p>ism of action underlying the neuroprotection</p><p>observed with niacin...</p></li></ul>