Liposome-entrapped superoxide dismutase reduces ischemia/reperfusion ‘oxidative stress’ in gerbil brain

Bilateral common carotid artery occlusion (15 min.) followed by two hours of recirculation reduced mitochondrial superoxide dismutase (SOD) and glutathione reductase (GR) activities, and increased susceptibility of mitochondrial membranes to in vitro lipid peroxidation in brain regions (i.e., cortex, striatum and hippocampus) of Mongolian gerbil. Intraperitoneal bolus injection (2 mg/kg b.w.) of liposome-entrapped CuZn superoxide dismutase (l-SOD) increased the endogenous SOD activity in normal brain tissue and, when given at the end of ischemia, counteracted both the ischemic reduction of endogenous SOD and the increased peroxidation of mitochondrial membranes. 1-SOD treatment was ineffective in reducing brain swelling, suggesting that superoxide radicals are not a main participant in the process of (post)ischemic brain edema formation.     

Modification of reperfusion-induced ionic imbalance by free radical scavengers in spontaneously hypertensive rat retina

We studied the effects of free radical scavengers, superoxide dismutase (SOD), vitamin E, and EGB 761, on ion shifts (Na⁺, K⁺, and Ca²⁺) induced by ischemia reperfusion in rat retina obtained from spontaneously hypertensive rats. Eyes were subjected to 90 min of retinal ischemia followed by 24 h of reperfusion. Two basic protocols were used: (1) chronic application, in which rats received SOD (7500, 15,000, and 30,000 U/kg, i.v.), vitamin E (50, 100, and 200 mg/kg, i.v.), and EGB 671 (50, 100, and 200 mg/kg, orally) for 10 d, respectively; and (2) acute administration, in which 7500, 15,000, and 30,000 U/kg of SOD, 50, 100, and 200 mg/kg of vitamin E, and 50, 100, and 200 mg/kg of EGB 761 were administered after an ischemic episode, at the onset of reperfusion, respectively. In the drug-free control group, 90 min ischemia followed by 24 h of reperfusion resulted in an accumulation of retinal sodium and calcium from their nonischemic control values of 76 ± 4 and 3.2 ± 0.1 μmol/g dry weight to 112 ± 6 (p < .001) and 6.2 (p < .001) μmol/g dry weight, respectively. Tissue potassium loss was also observed in this model of retinal ischemia reperfusion, and after 90 min ischemia followed by 24 h of reperfusion potassium content was significantly reduced from its nonischemic control value of 266 ± 5 to 207 ± 6 (p < .001) μmol/g dry weight. The chronic administration of SOD, vitamin E, and EGB 761 dose dependently reduced the reperfusion-induced ionic imbalance and improved the recovery of retinal ion contents. When these drugs were administered at the onset of reperfusion (acute administration), SOD and EGB 761 still significantly improved the recovery of retinal ion contents, but vitamin E failed to protect the ischemic reperfused retina. Our results indicate that the elimination of oxygen free radicals by free radicals scavengers may reduce the reperfusion-induced ionic imbalance and improve the ionic homeostasis in the injured retinal cells obtained from spontaneously hypertensive rats.     

Effect of intravenous eicosapentaenoic acid on cerebral blood flow, edema and brain prostaglandins in ischemic gerbils

Eicosapentaenoic acid is converted by cyclo-oxygenase to the prostacyclin, PGI3. Consequently eicosapentaenoic acid might protect the brain from the impairment in cerebral blood flow that follows temporary cerebral arterial occlusion. We studied the effect of 90% pure eicosapentaenoic acid, given intravenously, on cerebral blood flow, brain water and prostaglandins after ischemia in gerbils. Ischemia was produced by bilateral carotid occlusion for 15 min followed by reperfusion for 2 h. In experimental gerbils, 0.833 mg or 0.167 mg of eicosapentaenoic acid (Na salt) was given intravenously followed by a continuous infusion of 1 mg h-1. Control gerbils were given 0.167 mg of linoleic acid (Na salt) intravenously followed by a continuous infusion of 1 mg h-1 or a saline infusion. Regional cerebral blood flow was measured by the hydrogen clearance method and brain water by the specific gravity technique. Brain diene prostaglandins were measured by radioimmunoassay. In control gerbils cerebral blood flow decreased significantly during reperfusion and remained depressed after 2 h of reperfusion. In eicosapentaenoic acid treated gerbils blood flow decreased initially but after 2 h of reperfusion blood flow was significantly higher than in control gerbils. Brain edema and brain diene prostaglandins were not significantly different between control and experimental groups. Our study indicates that eicosapentaenoic acid, given intravenously, improves cerebral blood flow after ischemia and reperfusion. We speculate that this effect may be due to the formation of the prostacyclin, PGI3.     

Cerebroprotective activity of Wedelia calendulacea on global cerebral ischemia in rats

The present study was to investigate the effect of W. calendulacea on ischemia and reperfusion-induced cerebral injury. Cerebral ischemia was induced by occluding right and left common carotid arteries (global cerebral ischemia) for 30 min followed by reperfusion for 1 h and 4 h individually. Various biochemical alterations, produced subsequent to the application of bilateral carotid artery occlusion (BCAO) followed by reperfusion viz. increase in lipid peroxidation (LPO), hydrogen peroxide (H2O2), and decrease in reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD), level in the brain tissue, Western blot analysis (Cu-Zn-SOD and CAT) and assessment of cerebral infarct size were measured. All those enzymes are markedly reversed and restored to near normal level in the groups pretreated with W. calendulacea (250 and 500 mg/kg given orally in single and double dose/day for 10 days) in dose-dependent way. The effect of W. calendulacea had increased significantly the protein expression of copper/zinc superoxide dismutase (Cu-Zn-SOD) and CAT in cerebral ischemia. W. claendulacea was markedly decrease cerebral infarct damages but results are not statistically significant. It can be concluded that W. calendulacea possesses a neuroprotective activity against cerebral ischemia in rat.     

Superoxide dismutase,catalase, and U78517F attenuate neuronal damage in gerbils with repeated brief ischemic insults

Repeated ischemic insults at one hour intervals result in more severe neuronal damage than a single similar duration insult. The mechanism for the more severe damage with repetitive ischemia is not fully understood. We hypothesized that the prolonged reperfusion periods between the relatively short ischemic insults may result in a pronounced generation of oxygen free radicals (OFRs). In this study, we tested the protective effects of superoxide dismutase (SOD) and catalase (alone or in combination), and U78517F in a gerbil model of repetitive ischemia. Three episodes (two min each) of bilateral carotid occlusion were used at one hour intervals to produce repetitive ischemia. Superoxide dismutase and catalase were infused via osmotic pumps into the lateral ventricles. Two doses of U78517F were given three times per animal, one half hour prior to each occlusion. Neuronal damage was assessed 7 days later in several brain regions using the silver staining technique. The Mann-Whitney U test was used for statistical comparison. Superoxide dismutase showed significant protection in the hippocampus (CA4), striatum, thalamus and the medial geniculate nucleus (MGN). Catalase showed significant protection in the striatum, hippocampus, thalamus, and MGN and the substantia nigra reticulata. Combination of the two resulted in additional protection in the cerebral cortex. Compared to the controls, there was little protection with a dose of 3 mg/kg of U78517F. There was significant protection with a dose of 10 mg/kg in the hippocampus (CA4), striatum, thalamus, medial geniculate nucleus and the substantia nigra reticulata. The significant protection noted with SOD, catalase or U78517F with repeated ischemia supports, the hypothesis that OFRs may play a role in neuronal damage in repeated cerebral ischemia.     

MK-801 effect on regional cerebral oxidative stress rate induced by different duration of global ischemia in gerbils

We investigated MK-801 effect on ischemia-induced oxidative stress—the most important factor that exacerbates brain damage by reperfusion. The common carotid arteries of gerbils were occluded for 5, 10, or 15 min. Immediately after the occlusion, MK-801 (3 mg/kg i.p.) or saline were given in normothermic conditions. The MK-801 effects were followed in vivo by monitoring the neurological status of animals and at the intracellular level by standard biochemical assays. We investigated nitric oxide levels, superoxide production, superoxide dismutase activity, index of lipid peroxidation (ILP), and reduced glutathione content in hippocampus, striatum, forebrain cortex, and cerebellum. The measurements took place at different times (1, 2, 4, 7, 14, and 28 days) after reperfusion. Increased duration of cerebral ischemia resulted in a progressive induction of oxidative stress. Our results revealed pattern of dynamic changes in each oxidative stress parameter level which corresponded with ischemia duration in all tested brain structures. Most sensitive oxidative stress parameters were ILP and superoxide production. Our study confirmed spatial distribution of ischemia-induced oxidative stress. Tested brain structures showed different sensitivity to each oxidative stress parameter. As judged by biochemical and neurological data, applied MK-801 showed neuroprotective efficiency by reduction of ischemia-induced oxidative stress in brain.     

Chronic administration of ethyl docosahexaenoate decreases mortality and cerebral edema in ischemic gerbils

Dietary docosahexaenoic acid (DHA) intake can decrease the level of membrane arachidonic acid (AA), which is liberated during cerebral ischemia and implicated in the pathogenesis of brain damage. Therefore, in the present study, we investigated the effects of chronic ethyl docosahexaenoate (E-DHA) administration on mortality and cerebral edema induced by transient forebrain ischemia in gerbils. Male Mongolian gerbils were orally pretreated with either E-DHA (100, 150 mg/kg) or vehicle, once a day, for 4 weeks and were subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. The content of brain lipid AA at the termination of treatment, the survival ratio, change of regional cerebral blood flow (rCBF), brain free AA level, thromboxane B(2) (TXB(2)) production and cerebral edema formation following ischemia and reperfusion were evaluated. E-DHA (150 mg/kg) pretreatment significantly increased survival ratio, prevented post-ischemic hypoperfusion and attenuated cerebral edema after reperfusion compared with vehicle, which was well associated with the reduced levels of AA and TXB(2) in the E-DHA treated brain. These data suggest that the effects of E-DHA pretreatment on ischemic mortality and cerebral edema could be due to reduction of free AA liberation and accumulation, and its metabolite synthesis after ischemia and reperfusion by decreasing the content of membrane AA.     

Arachidonic Acid and Leukotriene C4: Role in Transient Cerebral Ischemia of Gerbils

Accumulation of arachidonic acid (AA) is greatest in brain regions most sensitive to transient ischemia. Free AA released after ischemia is either: 1) reincorporated into the membrane phospholipids, or 2) oxidized during reperfusion by lipoxygenases and cyclooxygenases, producing leukotrienes (LT), prostaglandins, thromboxanes and oxygen radicals. AA, its metabolite LTC₄ and lipid peroxides (generated during AA metabolism) have been implicated in the blood-brain barrier (BBB) dysfunction, edema and neuronal death after ischemia/reperfusion. This report describes the time course of AA release, LTC₄ accumulation and association with the physiological outcome during transient cerebral ischemia of gerbils. Significant amount of AA was detected immediately after 10 min ischemia (0 min reperfusion) which returned to sham levels within 30 min reperfusion. A later release of AA occurred after 1 d. LTC₄ levels were elevated at 0–6 h and 1 d after ischemia. Increased lipid peroxidation due to AA metabolism was observed between 2–6 h. BBB dysfunction occurred at 6 h. Significant edema developed at 1 and 2 d after ischemia and reached maximum at 3 d. Ischemia resulted in ~80% neuronal death in the CA₁ hippocampal region. Pretreatment with a 5-lipoxygenase inhibitor, AA861 resulted in significant attenuation of LTC₄ levels (Baskaya et al. 1996. J. Neurosurg. 85:112–116) and CA₁ neuronal death. Accumulation of AA and LTC₄, together with highly reactive oxygen radicals and lipid peroxides, may alter membrane permeability, resulting in BBB dysfunction, edema and ultimately to neuronal death.     

‘Therapeutic window’ for multiple drug treatment of experimental cerebral ischemia in gerbils

The effects of the following drugs: nimodipine (1 mg/kg b. w., i. p.), 2-amino-5-phosphonovaleric acid (4mg/kg b.w., i.p.) and propentofylline (25mg/kg b.w., i.p.), administered (alone or in combination) at the end of 15 min bilateral ischemia in gerbils were evaluated on mitochondrial superoxide dismutase (SOD), glutathione reductase (GR), glucose-6 phosphate dehydrogenase (G6PD), monoamine oxidase (MAO) activities, and thiobarbituric acid reactive material (TBARM), and brain water content at 1 hour of reperfusion. The combined treatment virtually abolished early postischemic brain edema (4.1% v.s. 0.6%) and efficiently counteracted ischemia-induced changes [decreased SOD (79% v.s. 98%), GR (52% v.s. 105%) and MAO (25% v.s. 79%), and increased TBARM (198% v.s. 108%)]. The same combination of drugs administered 15 min before ischemia had a similar effect (e.g., reduced brain swelling and lipid peroxidation) as when given at the end of ischemia, whereas a limited or absent impact was seen when the drugs were given 15 min or 1 hour after ischemia, respectively. The data suggest that (post)ischemic brain swelling and mitochondrial dysfunction can be reduced by drugs which synchronously prevent processes induced in the early stages of reperfusion.     

Effect of intravenous eicosapentaenoic acid on cerebral blood flow, edema and brain prostaglandins in ischemic gerbils

Eicosapentaenoic acid is converted by cyclo-oxygenase to the prostacyclin, PGI₃. Consequently eicosapentaenoic acid might protect the brain from the impairment in cerebral blood flow that follows temporary cerebral artirial occlusion. We studied the effect of 90% pure eicosapentaenoic acid, given intravenously, on cerebral blood flow, brain water and prostaglandins after ischemia in gerbils. Ischemia was produced by bilateral carotid occlusion for 15 min followed by reperfusion for 2 h. In experimental gerbils, 0.833 mg or 0.167 mg of eicosapentaenoic acid (Na salt) was given intravenously followed by a continuous infusion of 1 mg h⁻¹. Control gerbils were given 0.167 mg of linoleic acid (Na salt) intravenously followed by a continuous infusion of 1 mg h⁻¹ or a saline infusion. Regional cerebral blood flow was measured by the hydrogen clearance method and brain water by the specific gravity technique. Brain diene prostaglandins were measured by radioimmunoassay. In control gerbils cerebral blood flow decreased significantly during reperfusion and remained depressed after 2 h of reperfusion. In eicosapentaenoic acid treated gerbils blood flow decreased initially but after 2 h of reperfusion blood flow was significantly higher than in control gerbils. Brain edema and brain diene prostaglandins were not significantly different between control and experimental groups.Our study indicates that eicosapentaenoic acid, given intravenously, improves cerebral blood flow after ischemia and reperfusion. We speculate that this effect may be due to the formation of the prostacyclin, PGI₃.     

Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. Several in vitro and in vivo studies have proved the antioxidant potential of curcumin and its metabolites. Hence, in the present study the neuroprotective potential of curcumin was investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral IR injury. 2 h of MCAO and 22 h of reperfusion resulted in the infarct volume of 210.39 +/- 31.25 mm3. Administration of curcumin 100 and 300 mg/kg, i.p. 30 min. after MCAO produced 37.23 +/- 5.10% and 46.39 +/- 10.23% (p < 0.05) reduction in infarct volume, respectively. Ischemia induced cerebral edema was reduced in a dose dependent manner. Curcumin at 300 mg/kg, i.p. produced 50.96 +/- 6.04% reduction in edema (p < 0.05) volume. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed, which was reduced by curcumin (300 mg/kg, i.p.)-treatment. Decrease in superoxide dismutase and glutathione peroxidase activity was observed in ipsilateral hemisphere of MCAO animal. Curcumin-treatment (300 mg/kg, i.p.) prevented IR injury mediated fall in glutathione peroxide activity. Peroxynitrite measured using rhodamine123 fluorescence and anti-nitrotyrosine immunofluorescence indicated increased peroxynitrite formation after IR insult. Curcumin-treatment reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest the neuroprotective potential of curcumin in cerebral ischemia and is mediated through its antioxidant activity.     

Chronic administration of ethyl docosahexaenoate reduces gerbil brain eicosanoid productions following ischemia and reperfusion

Arachidonic acid (AA) and its vasoactive metabolites have been implicated in the pathogenesis of brain damage induced by cerebral ischemia. The membrane AA concentrations can be reduced by changes in dietary fatty acid intake. The purpose of the present study was to investigate the effects of chronic ethyl docosahexaenoate (E-DHA) administration on the generation of eicosanoids of AA metabolism during the period of reperfusion after ischemia in gerbils. Weanling male gerbils were orally pretreated with either E-DHA (100, 200 mg/kg) or vehicle, once a day, for 10 weeks, and subjected to transient forebrain ischemia by bilateral common carotid occlusion for 10 min. E-DHA (200 mg/kg) pretreatment significantly decreased the content of brain lipid AA at the termination of treatment, prevented postischemic impaired regional cerebral blood flow (rCBF) and reduced the levels of brain prostaglandin (PG) PGF(2alpha) and 6-keto-PGF(1alpha), and thromboxane B(2) (TXB(2)), as well as leukotriene (LT) LTB(4) and LTC(4) at 30 and 60 min of reperfusion compared with the vehicle, which was well associated with the attenuated cerebral edema in the E-DHA-treated brain after 48 h of reperfusion. These data suggest that the E-DHA (200 mg/kg) pretreatment reduces the postischemic eicosanoid productions, which may be due to its reduction of the brain lipid AA content.     

A new approach for extracellular spin trapping of nitroglycerin-induced superoxide radicals both in vitro and in vivo

Anti-ischemic therapy with nitrates is complicated by the induction of tolerance that potentially results from an unwanted coproduction of superoxide radicals. Therefore, we analyzed the localization of in vitro and in vivo, glyceryl trinitrate (GTN)-induced formation of superoxide radicals and the effect of the antioxidant vitamin C and of superoxide dismutase (SOD). Sterically hindered hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidin (PP-H) can be used for in vitro and in vivo quantification of superoxide radical formation. The penetration/incorporation of CP-H or PP-H and of their corresponding nitroxyl radicals was examined by fractionation of the blood and blood cells during a 1-h incubation. For monitoring in vivo, GTN-induced (130 microg/kg) O2*- formation CP-H or PP-H were continuously infused (actual concentration, 800 microM) for 90 to 120 min into rabbits. Formation of superoxide was determined by SOD- or vitamin C-inhibited contents of nitroxide radicals in the blood from A. carotis. The incubation of whole blood with CP-H, PP-H, or corresponding nitroxyl radicals clearly shows that during a 1-h incubation, as much as 8.3% of CP-H but only 0.9% of PP-H is incorporated in cytoplasm. Acute GTN treatment of whole blood and in vivo bolus infusion significantly increased superoxide radical formation as much as 4-fold. Pretreatment with 20 mg/kg vitamin C or 15,000 U/kg superoxide dismutase prevented GTN-induced nitroxide formation. The decrease of trapped radicals after treatment with extracellularly added superoxide dismutase or vitamin C leads to the conclusion that GTN increases the amount of extracellular superoxide radicals both in vitro and in vivo.     

Evaluation of antioxidant and neuroprotective effect of date palm (Phoenix dactylifera L.) against bilateral common carotid artery occlusion in rats

The cerebral ischemia in rats was induced by occluding bilateral common carotid arteries (BCCAO) for 30 min., followed by 45 min reperfusion. BCCAO caused significant depletion in superoxide dismutase, catalase, glutathione, glutathione peroxidase, glutathione-S-transferase, glutathione reductase and significant increase in lipid peroxidation along with severe neuronal damage in the brain. All the alterations except depletion in glutathione peroxidase and glutathione-S-transferase levels induced by cerebral ischemia were significantly attenuated by 15 days pretreatment with methanolic extract of P. dactylifera fruits (100, 300 mg/kg), whereas 30 mg/kg dose was insignificant in this regard. These results suggest the possible use P. dactylifera against bilateral common carotid artery occlusion induced oxidative stress and neuronal damage.     

Possible neuroprotective effects of lecithin and alpha-tocopherol alone or in combination against ischemia/reperfusion insult in rat brain

A close correlation exists between ischemia/reperfusion (I/R)-induced insult and the release of free radicals. Lecithin is a polyunsaturated phosphatidylcholine that corresponds to the phosphatidylcholine molecule. Phosphatidylcholines are high-energy functional and structural elements of all biologic membranes. alpha-Tocopherol is the major lipid-soluble chain-breaking antioxidant in the body tissues and effectively protects against neuronal damage. Therefore, we studied the effect of lecithin (300 mg/kg, p.o., 14 days) and alpha-tocopherol (200 mg/kg, p.o., 14 days), alone or in combination, on the brain redox state during I/R. Adult male Wistar rats were subjected to global ischemia by the occlusion of the two carotid arteries 24 h after the last dose of drug administration. Reperfusion was carried out 1 h after induction of ischemia and lasted for another hour. Brain lipid peroxides (MDA) and glutathione (GSH) contents, as well as superoxide dismutase (SOD) and catalase (CAT) activities were assessed. The results showed that I/R elevated brain lipid peroxides content which was accompanied by a reduction in both antioxidant enzyme activities, however, brain GSH level remained unaltered. Lecithin, alpha-tocopherol and their combination restored MDA content, as well as CAT activity with a slight tendency to normalize SOD activity. We conclude that lecithin has a possible neuroprotective effect partly through its antioxidant action which is comparable to that of alpha-tocopherol.     

Recombinant human superoxide dismutase can attenuate ischemic neuronal damage in gerbils.

The effects of recombinant human superoxide dismutase (r-hSOD) on ischemic neuronal injury were examined. Cerebral ischemia was produced in Mongolian gerbils by occluding bilateral common carotid arteries for 5 min. Preischemic treatment with r-hSOD clearly reduced hippocampal neuronal damages while postischemic treatment did not. This result suggests that oxygen free radicals play an important role in selective vulnerability to ischemia and r-hSOD has a potential clinical usefulness against cerebral ischemia.     

Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils

The beneficial effects of antioxidant nutrients, as well as complex plant extracts, in cerebral ischemia/reperfusion brain injury are well known. Mediterranean diet, rich in olive products, is associated with lower incidence of cardiovascular disease, cancer, inflammation and stroke. In this study, the possible neuroprotective effect of standardized dry olive leaf extract (OLE) is investigated for the first time. Transient global cerebral ischemia in Mongolian gerbils was used to investigate the OLE effects on different parameters of oxidative stress and neuronal damage in hippocampus. The biochemical measurements took place at different time points (80 min, 2, 4 and 24 h) after reperfusion. The effects of applied OLE were compared with effects of quercetin, a known neuroprotective plant flavonoid. Pretreatment with OLE (100 mg/kg, per os) significantly inhibited production of superoxide and nitric oxide, decreased lipid peroxidation, and increased superoxide dismutase activity in all time points examined. Furthermore, OLE offered histological improvement as seen by decreasing neuronal damage in CA1 region of hippocampus. The effects of applied OLE were significantly higher than effects of quercetin (100 mg/kg, per os). Our results indicate that OLE exerts a potent neuroprotective activity against neuronal damage in hippocampus after transient global cerebral ischemia, which could be attributed to its antioxidative properties.     

Neuroprotective potential of group I metabotropic glutamate receptor antagonists in two ischemic models

The neuroprotective potential of mGluR1 and mGluR5 antagonists (group I), EMQMCM and MTEP, respectively was studied using the 3 min forebrain ischemia model in Mongolian gerbils and the hypoxia-ischemia model in 7-day-old rats. Hypoxia-ischemia was induced by unilateral carotid occlusion followed by 75 min exposure to hypoxia (7.3% O(2) in N(2)), forebrain ischemia in gerbils was evoked by bilateral common carotid artery occlusion. The postischemic rectal body temperature in rat pups or brain temperature of gerbils was measured. The drugs were administered i.p. three times every 2 h after the insult, each time in equal doses of 1.25, 2.5 or 5.0 mg/kg. After 2 weeks brain damage was evaluated as weight decrease of the ipsilateral hemisphere in the rat pups or damage to CA1 pyramids in the gerbil hippocampus. The results demonstrated a dose dependent neuroprotection in both ischemic models by EMQMCM, while MTEP was neuroprotective only in the gerbil model of forebrain ischemia. EMQMCM reduced postischemic hyperthermia in gerbils. Thus, the antagonists of mGluR1 and mGluR5 show differential neuroprotective ability in two models of brain ischemia. Postischemic hypothermia may be partially involved in the mechanism of neuroprotection following EMQMCM in gerbils.     

Changes of phospholipid composition and superoxide dismutase activity during global brain ischemia and reperfusion in rats

Alterations in phospholipid content and Cu/Zn superoxide dismutase (SOD) activity were examined in rat brain after 15 min of global ischemia (four-vessel occlusion) followed by 2-, 24- or 48-h reperfusion. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the main brain phospholipids, were markedly decreased in ischemic rats and remained decreased during the whole reperfusion period. Concentrations of phosphatidylinositol (PI) and sphingomyelin (SM) were also significantly reduced during ischemia but recovered during reperfusion period. In contrast, phosphatidylserine (PS) and lysophospholipids (LysoPL) were unchanged during ischemia but were elevated after 24 h of reperfusion. Significant reductions in blood plasma phospholipids were also demonstrated. 24-48 h of reperfusion markedly decreased PE, PC and PS contents, while the concentrations were almost unchanged by ischemia alone. Brain SOD activity decreased significantly during ischemia and was recovered to control value already after 2 h of reperfusion. These results suggest that ischemia/reperfusion is accompanied by a significant and selective degradation of brain phospholipids that may be attributable to oxidative stress and activation of phospholipases.     

Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury

There has been considerable controversy regarding the role of oxygen free radicals as important mediators of cell damage in reperfused myocardium. This controversy regards whether superoxide and hydroxyl free radicals are generated on reperfusion and if these radicals actually cause impaired contractile function. In this study, EPR studies using the spin trap 5,5-dimethyl-1-pyroline-n-oxide (DMPO) demonstrate the formation of .OH and R. free radicals in the reperfused heart. EPR signals of DMPO-OH, aN = aH = 14.9 G, and DMPO-R aN = 15.8 G aH = 22.8 G are observed, with peak concentrations during the first minute of reperfusion. It is demonstrated that these radicals are derived from .O2- since reperfusion in the presence of enzymatically active recombinant human superoxide dismutase markedly reduced the formation of these signals while inactive recombinant human superoxide dismutase had no effect. On reperfusion with perfusate pretreated to remove adventitial iron, the concentration of the DMPO-OH signal was increased 2-fold and a 4-fold decrease in the DMPO-R signal was observed demonstrating that iron-mediated Fenton chemistry occurs. Hearts reperfused with recombinant human superoxide dismutase exhibited improved contractile function in parallel with the marked reduction in measured free radicals. In order to determine if the reperfusion free radical burst results in impaired contractile function, simultaneous measurements of free radical generation and contractile function were performed. A direct relationship between free radical generation and subsequent impaired contractile function was observed. These studies suggest that superoxide derived .OH and R. free radicals are generated in the reperfused heart via Fenton chemistry. These radicals appear to be key mediators of myocardial reperfusion injury.