Huperzine a restores cortico-hippocampal functional connectivity after bilateral AMPA lesion of the nucleus basalis of meynert.

Huperzine A (Hup-A), an alkaloid isolated from Huperzia serrata (Thunb.) Trevis. (Lycopodiaceae), acts as a selective inhibitor of acetylcholinesterase and shows memory-enhancing properties. Although Hup-A has shown promising expectation for Alzheimer's disease (AD) patients, controlled clinical trials supporting its use are limited. The aim of this work was to study in vivo, in an animal model of AD, the pharmacological activity of systemic administration of Hup-A on cortex- and hippocampus-dependent memory. With this purpose, a set of experiments was planned to evaluate attention, learning, working and spatial memory with respect to cortical and hippocampal electroencephalogram (EEG) theta rhythm during the object recognition test and Morris water maze in animals with lesion of the nucleus basalis of Meynert (NBM). In NBM-lesioned animals, compared with control, an increased theta power in the cortex and a reduced theta rhythm oscillation in the hippocampus were found. These EEG changes were correlated with worse performance in learning and memory tasks. In rats with damaged NBM, Hup-A (0.5 mg/kg i.p.) was able to restore EEG architecture, producing cortical desynchronization and reduction in theta power, while in the hippocampus the drug increased theta oscillation and reduced the impairment in attention/working memory as well as spatial navigation performance in the behavioral tasks. Taken together, the present data suggest that Hup-A is able to restore cholinergic cortico-hippocampal functional connectivity. In conclusion, the present results are in agreement with other experimental evidence that promote the clinical use of this natural drug.

The antidote effect of quinone oxidoreductase 2 inhibitor against paraquat-induced toxicity in vitro and in vivo.

BACKGROUND AND PURPOSE The mechanisms of paraquat (PQ)-induced toxicity are poorly understood and PQ poisoning is often fatal due to a lack of effective antidotes. In this study we report the effects of N-[2-(2-methoxy-6H-dipyrido{2,3-a:3,2-e}pyrrolizin-11-yl)ethyl]-2-furamide (NMDPEF), a melatonin-related inhibitor of quinone oxidoreductase2 (QR2) on the toxicity of PQ in vitro & in vivo. EXPERIMENTAL APPROACH Prevention of PQ-induced toxicity was tested in different cells, including primary pneumocytes and astroglial U373 cells. Cell death and reactive oxygen species (ROS) were analysed by flow cytometry and fluorescent probes. QR2 silencing was achieved by lentiviral shRNAs. PQ (30 mg·kg(-1)) and NMDPEF were administered i.p. to Wistar rats and animals were monitored for 28 days. PQ toxicity in the substantia nigra (SN) was tested by a localized microinfusion and electrocorticography. QR2 activity was measured by fluorimetry of N-benzyldihydronicotinamide oxidation. KEY RESULTS NMDPEF potently antagonized non-apoptotic PQ-induced cell death, ROS generation and inhibited cellular QR2 activity. In contrast, the cytoprotective effect of melatonin and apocynin was limited and transient compared with NMDPEF. Silencing of QR2 attenuated PQ-induced cell death and reduced the efficacy of NMDPEF. Significantly, NMDPEF (4.5 mg·kg(-1)) potently antagonized PQ-induced systemic toxicity and animal mortality. Microinfusion of NMDPEF into SN prevented severe behavioural and electrocortical effects of PQ which correlated with inhibition of malondialdehyde accumulation in cells and tissues. CONCLUSIONS AND IMPLICATIONS NMDPEF protected against PQ-induced toxicity in vitro and in vivo, suggesting a key role for QR2 in the regulation of oxidative stress.

Toxic profile of bergamot essential oil on survival and proliferation of SH-SY5Y neuroblastoma cells.

Cosmetic, pharmaceutical, food and confectionary industries make increasing use of plant extracts in their products. Despite the widespread use of products containing plant extracts, the mechanisms of their effects are not fully characterized. Bergamot essential oil (BEO; Citrus bergamia, Risso) is a well-known plant extract used in aromatherapy and it has analgesic, anxiolytic and neuroprotective effects in rodents. To elicit neuroprotection, BEO recruits Akt prosurvival pathways. However, Akt stimulates cell proliferation, which may also pose risks for health in case of prolonged use. To study the potential effects of BEO on survival and proliferation of dividing cells, we selected human SH-SY5Y neuroblastoma cells. BEO triggered concentration-dependent mitochondrial dysfunction, cytoskeletal reorganization, cell shrinkage, DNA fragmentation and both caspase-dependent and independent cell death. Analysis of cleavage products of poly-(ADP-ribose) polymerase (PARP) revealed caspase-3 activation, but also activation of additional protease families. As result of increased proteolytic activity, Akt protein levels decreased in BEO-treated cells. Our data show that BEO can be lethal for dividing cells by activating multiple pathways. While this may reduce the risk of unwanted cell proliferation after prolonged use, it does suggest a cautionary approach to the use of inappropriate dilutions of the oil that may cause cell death.

Hypolipemic and hypoglycaemic activity of bergamot polyphenols: from animal models to human studies.

Bergamot juice produces hypolipemic activity in rats though the mechanism remains unclear. Here we investigated on the effect of bergamot extract (BPF) in diet-induced hyperlipemia in Wistar rats and in 237 patients suffering from hyperlipemia either associated or not with hyperglycaemia. BPF, given orally for 30 days to both rats and patients, reduces total and LDL cholesterol levels (an effect accompanied by elevation of cHDL), triglyceride levels and by a significant decrease in blood glucose. Moreover, BPF inhibited HMG-CoA reductase activity and enhanced reactive vasodilation thus representing an efficient phytotherapeutic approach in combating hyperlipemic and hyperglycaemic disorders.

The protective effect of tianeptine on Gp120-induced apoptosis in astroglial cells: role of GS and NOS, and NF-κB suppression.

BACKGROUND AND PURPOSE: Tianeptine is an antidepressant affecting the glutamatergic system. In spite of its proven clinical efficacy, molecular effects of tianeptine are not entirely clear. Tianeptine modulates cytokine expression in the CNS and protects the hippocampus from chronic stress effects. HIV infection is associated with inflammation and neuronal loss, causing HIV-associated dementia (HAD). The human immunodeficiency virus type-1 glycoprotein gp120 has been proposed as a likely aetiological agent of HAD. In this study, we determined whether tianeptine protects astroglial cells from the neurodegenerative effects of gp120. EXPERIMENTAL APPROACH: Human astroglial cells were treated with gp120 and tianeptine, and viability and apoptosis was monitored by TUNEL, annexin V, and activated caspase-3 staining and flow cytometry. Protein levels of glutamine synthase (GS), inducible and constitutive nitric oxide synthases (iNOS, cNOS) and nuclear factor κB (NF-κB) pathway were determined by Western blot analysis. The respective activities were assessed indirectly by measuring glutamine and nitrite concentrations or by luciferase reporter assays. KEY RESULTS: Tianeptine showed an anti-apoptotic effect and prevented caspase-3 activation by gp120. The mechanism of tianeptine's action involved GS and cNOS stabilization and iNOS suppression. Moreover, tianeptine increased IκB-α levels in the absence of gp120 and blocked its degradation in response to gp120. This correlated with the suppression of basal and gp120-induced NF-κB transcriptional activity. CONCLUSIONS AND IMPLICATIONS: Tianeptine clearly exerts neuroprotective effects in vitro by suppressing the molecular pro-inflammatory effects of gp120. Studies in animal models should be performed to evaluate the potential of tianeptine as a treatment for HAD.

Simple choline esters as potential anti-Alzheimer agents.

In this manuscript we report an integrated study to develop simple choline esters as cholinergic agents potentially useful against the Alzheimer disease. In previously reported experiments we demonstrated the capability of the pivaloylcholine to exert cholinergic effects into the Central Nervous System, so we decided to explore small variants of choline esters. The knowledge of crystallographic models of the enzymes involved in the hydrolysis of the acetylcholine allowed to consider the steric compatibility of some choline derivatives within their catalytic sites. The purpose of the work was to find out analogues with increased selectivity toward the acetylcholinesterase versus the butyrrylcholinesterase. Theoretical models were compared to enzymatic tests carried out with both enzymes and two different methods. In this screening we have selected two candidates for the in vivo experiments with pre-treated rats. Their electroencephalographic profiles were recorded and averaged before and after the intraperitoneal treatment with two compounds in comparison to the pivaloyl lead ester. The results demonstrated that one of the esters can exert biological effects similar to the parent compound.

Neuropharmacology of the essential oil of bergamot.

Bergamot (Citrus bergamia, Risso) is a fruit most knowledgeable for its essential oil (BEO) used in aromatherapy to minimize symptoms of stress-induced anxiety and mild mood disorders and cancer pain though the rational basis for such applications awaits to be discovered. The behavioural and EEG spectrum power effects of BEO correlate well with its exocytotic and carrier-mediated release of discrete amino acids endowed with neurotransmitter function in the mammalian hippocampus supporting the deduction that BEO is able to interfere with normal and pathological synaptic plasticity. The observed neuroprotection in the course of experimental brain ischemia and pain does support this view. In conclusion, the data yielded so far contribute to our understanding of the mode of action of this phytocomplex on nerve tissue under normal and pathological experimental conditions and provide a rational basis for the practical use of BEO in complementary medicine. The opening of a wide venue for future research and translation into clinical settings is also envisaged.

A novel animal model to evaluate the ability of a drug delivery system to promote the passage through the BBB.

The purpose of this investigation was to explore the potentiality of a novel animal model to be used for the in vivo evaluation of the ability of a drug delivery system to promote the passage through the blood-brain barrier (BBB) and/or to improve the brain localization of a bioactive compound. A Tween 80-coated poly-L-lactid acid nanoparticles was used as a model of colloidal drug delivery system, able to trespass the BBB. Tacrine, administered in LiCl pre-treated rats, induces electrocorticographic seizures and delayed hippocampal damage. The toxic effects of tacrine-loaded poly-L-lactid acid nanoparticles (5mg/kg), a saline solution of tacrine (5mg/kg) and an empty colloidal nanoparticle suspension were compared following i.p. administration in LiCl-pre-treated Wistar rats. All the animals treated with tacrine-loaded nanoparticles showed an earlier outcome of CNS adverse symptoms, i.e. epileptic onset, with respect to those animals treated with the free compound (10 min vs. 22 min respectively). In addition, tacrine-loaded nanoparticles administration induced damage of neuronal cells in CA1 field of the hippocampus in all treated animals, while the saline solution of tacrine only in 60% of animals. Empty nanoparticles provided similar results to control (saline-treated) group of animals. In conclusion, the evaluation of time-to-onset of symptoms and the severity of neurodegenerative processes induced by the tacrine-lithium model of epilepsy in the rat, could be used to evaluate preliminarily the capability of a drug delivery system to trespass (or not) the BBB in vivo.

Effects of systemic administration of the essential oil of bergamot (BEO) on gross behaviour and EEG power spectra recorded from the rat hippocampus and cerebral cortex.

Bergamot (Citrus bergamia Risso et Poiteau) is a citrus fruit growing almost exclusively in the South of Italy. Its essential oil is obtained by cold pressing of the epicarp and, partly, of the mesocarp of the fresh fruit. Although this phytocomplex has been used for centuries, reputedly effectively, as a traditional medicine, there is very little verified scientific evidence to support this use. This paper reports original data on the systemic effects of the essential oil of bergamot (BEO) on gross behaviour and EEG activity recorded from the hippocampus and cerebral cortex of the rat. The Fast Fourier Transformation (FFT) was used to analyse and quantify the energy in single frequency bands of the EEG spectrum. The results obtained indicate that systemic administration of increasing volumes of BEO produces dose-dependent increases in locomotor and exploratory activity that correlate with a predominant increase in the energy in the faster frequency bands of the EEG spectrum. These data contribute to our understanding of the neurobiological profile of BEO.

Prevention of Glutamate Accumulation and Upregulation of Phospho-Akt may Account for Neuroprotection Afforded by Bergamot Essential Oil against Brain Injury Induced by Focal Cerebral Ischemia in Rat.

The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on brain damage caused by permanent focal cerebral ischemia in rat were investigated. Administration of BEO (0.1-0.5 ml/kg but not 1 ml/kg, given intraperitoneally 1 h before occlusion of the middle cerebral artery, MCAo) significantly reduced infarct size after 24 h permanent MCAo. The most effective dose (0.5 ml/kg) resulted in a significant reduction of infarct extension throughout the brain, especially in the medial striatum and the motor cortex as revealed by TTC staining of tissue slices. Microdialysis experiments show that BEO (0.5 ml/kg) did not affect basal amino acid levels, whereas it significantly reduced excitatory amino acid, namely aspartate and glutamate, efflux in the frontoparietal cortex typically observed following MCAo. Western blotting experiments demonstrated that these early effects were associated, 24 h after permanent MCAo, to a significant increase in the phosphorylation and activity of the prosurvival kinase, Akt. Indeed, BEO significantly enhanced the phosphorylation of the deleterious downstream kinase, GSK-3beta, whose activity is negatively regulated via phosphorylation by Akt.

Identification of novel pharmacological targets to minimize excitotoxic retinal damage.

Excitotoxic neuronal death is a common feature of neurodegenerative and ischemic diseases of the central nervous system (CNS) and of a variety of ocular diseases, including glaucoma. Glaucoma, one of the leading causes of blindness in the world, is characterized by a progressive degeneration of retinal ganglion cells (RGCs) and their axons and is often associated with elevated intraocular pressure (IOP). Retinal ischemia/reperfusion induced by experimental elevation of IOP leads to damage and loss of RGCs. Under these conditions, structural, functional, and biochemical changes implicate the accumulation of extracellular glutamate and activation of the excitotoxic cascade. Beside the activation of associated pathways, death of RGCs is accompanied by impaired endogenous defenses, such as the PI3K/Akt prosurvival pathway. Original neurochemical and pharmacological evidence are discussed here to strengthen the role for excitotoxicity in RGCs death occurring in experimental, angle closure, glaucoma in conjunction with the discovery of novel molecular targets to potentiate endogenous prosurvival defenses in the glaucomatous retina.

The protective effect of bergamot oil extract on lecitine-like oxyLDL receptor-1 expression in balloon injury-related neointima formation.

Lectin-like oxyLDL receptor-1 (LOX-1) has recently been suggested to be involved in smooth muscle cell (SMC) proliferation and neointima formation in injured blood vessels. This study evaluates the effect of the nonvolatile fraction (NVF), the antioxidant component of bergamot essential oil (BEO), on LOX-1 expression and free radical generation in a model of rat angioplasty. Common carotid arteries injured by balloon angioplasty were removed after 14 days for histopathological, biochemical, and immunohistochemical studies. Balloon injury led to a significant restenosis with SMC proliferation and neointima formation, accompanied by increased expression of LOX-1 receptor, malondialdehyde and superoxide formation, and nitrotyrosine staining. Pretreatment of rats with BEO-NVF reduced the neointima proliferation together with free radical formation and LOX-1 expression in a dose-dependent manner. These results suggest that natural antioxidants may be relevant in the treatment of vascular disorders in which proliferation of SMCs and oxyLDL-related endothelial cell dysfunction are involved.

Choline pivaloyl ester enhances brain expression of both nerve growth factor and high-affinity receptor TrkA, and reverses memory and cognitive deficits, in rats with excitotoxic lesion of nucleus basalis magnocellularis.

The aim of present work was the evaluation of the effects on brain levels of nerve growth factor (NGF) and of its high-affinity tyrosine kinase A receptor (TrkA), induced in rats unilaterally lesioned at nucleus basalis magnocellularis (NBM), by treatment with choline pivaloyl ester (CPE). CPE was daily administered (60 micromol/Kg ip) during 3 weeks to rats selectively lesioned by AMPA infusion into right NBM; the intact left NBM serving as control. NGF levels were determined in cerebral cortex and hippocampus by Elisa assay. TrkA receptor expression was evaluated in right NBM by Western blotting analysis. CPE treatment significantly increased NGF levels in both hippocampus and neocortex in right NBM, compared with intact left counter-part and controls. Western blotting showed an evident enhancement in TrkA receptor expression in lesioned right NBM in comparison with intact left counter-part and controls. CPE treatment was also able to restore, in bilaterally NBM-lesioned rats, the disrupted cortical EEG and HVS activities as well as to reverse deficits in learning and memory in spatial navigation and probe trials, and cognitive capacities in object recognition task.

N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction.

BACKGROUND: HIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC). Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood. Here we investigated on the effect of N-acetylcysteine (NAC), on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS) in the imbalanced activity of glutamine synthase (GS), the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders. RESULTS: Incubation of Lipari human cultured astroglial cells with gp 120 (0.1-10 nM) produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS). This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA). In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5-5 mM), dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells. CONCLUSION: In conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered glutamine release. All the effects of gp120 on astroglial cells were counteracted by NAC thus suggesting a novel and potentially useful approach in the treatment of glutammatergic disorders found in HAD patients.

Electrocortical effects of MDMA are potentiated by acoustic stimulation in rats.

BACKGROUND: 3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is known for its toxicological, psychopathological and abuse potential. Some environmental conditions, e.g. acoustic stimulation typical of the "rave scene" can influence the toxicity of this drug. RESULTS: We investigated the effects of low doses of MDMA in vivo using Wistar rats in the absence of acoustic stimulation (white noise; 95 Db) demonstrating that ecstasy is able to induce a significant activation (reduction of Electrocortical total power) of the telencephalic cortex that spontaneously reverts in the absence of sensorial stimuli, whereas it persists for several days if, in addition to MDMA, the animals are exposed to acoustic stimulation. CONCLUSION: Our data demonstrate that low doses of MDMA are able to reduce electrocortical total power, and that this effect is potentiated by sensorial stimuli commonly present in certain environments, such as rave parties.

Endothelial nitric oxide synthase is segregated from caveolin-1 and localizes to the leading edge of migrating cells.

The enzyme endothelial Nitric Oxide Synthase (eNOS) is involved in key physiological and pathological processes, including cell motility and apoptosis. It is widely believed that at the cell surface eNOS is localized in caveolae, where caveolin-1 negatively regulates its activity, however, there are still uncertainties on its intracellular distribution. Here, we applied high resolution confocal microscopy to investigate the surface distribution of eNOS in transfected HeLa cells and in human umbilical vein endothelial cells (HUVEC) endogenously expressing the enzyme. In confluent and non-confluent HUVEC and HeLa cells, we failed to detect substantial colocalization between eNOS and caveolin-1 at the cell surface. Instead, in non-confluent cells, eNOS was concentrated in ruffles and at the leading edge of migrating cells, colocalizing with actin filaments and with the raft marker ganglioside G(M1), and well segregated from caveolin-1, which was restricted to the posterior region of the cells. Treatments that disrupted microfilaments caused loss of eNOS from the cell surface and decreased Ca(2+)-stimulated activity, suggesting a role of the cytoskeleton in the localization and function of the enzyme. Our results provide a morphological correlate for the role of eNOS in cell migration and raise questions on the site of interaction between eNOS and caveolin-1.

Amino acid levels in some lethargic mouse brain areas before and after pentylenetetrazole kindling.

Genetic animal models have contributed significantly to our understanding of epilepsy causes. Lethargic mice are considered a valid model of absence epilepsy, which have been shown to possess behavioral, electrographic and pharmacological profiles similar to those of humans with absence epilepsies. Single gene mutations that comprise the beta4 subunit of voltage-sensitive Ca2+ channels underlie the spontaneous discharges of the absence, non-convulsive seizures of lethargic mice. There are no available data concerning how the mutant channels actually behave at terminals in response to chemical activation by subconvulsant stimulation with pentylenetetrazole. In this study, we found no significant difference in the convulsive dose 50 between lethargic and control mice. Lethargic mice showed a more rapid development of kindling to pentylenetetrazole than control animals. No significant differences were observed between the groups of mice rechallenged with pentylenetetrazole 30 or 60 days after the end of the chronic treatment. Marked differences in brain amino acid levels were found between the two strains of mice in basal conditions and after kindling. In conclusion, our results indicate that lethargic mice show a range of biochemical and behavioral changes, correlated in particular with a higher susceptibility to develop kindled seizures.

17beta-estradiol protects SH-SY5Y Cells against HIV-1 gp120-induced cell death: evidence for a role of estrogen receptors.

Despite the large body of experimental evidence demonstrating the neuroprotective properties of 17beta-estradiol (17beta-E2) both in vitro and in vivo experimental models of neuronal injury, the exact mechanisms implicated in neuroprotection have not been fully delineated. Some experimental evidence highlight a role for the antioxidant properties of 17beta-E2 in mediating protection against oxidative injury. Parallel to these, evidence also exist which point to alternative mechanisms involving estrogen receptors (ER). The HIV-1 coat protein, gp120, has been implicated in the progression of central nervous system damage caused by HIV-1 infection. The neurotoxic effects induced by gp120 are triggered via an excitotoxic mechanism of cell death which implicates alteration of calcium homeostasis, activation of calcium-dependent pathways, mitochondrial uncoupling and membrane lipid peroxidation. In the present study, we demonstrate that 17beta-E2 protects human SH-SY5Y neuroblastoma cells from cell death elicited by gp120. Tamoxifen and ICI 182,780, two ER antagonists, both antagonized 17beta-E2-mediated inhibition of cell death. Exposure of SH-SY5Y cells to gp120 for 30min caused a significant accumulation of intracellular reactive oxygen species (ROS) and this was abrogated by 17beta-E2; however, the ability of 17beta-E2 to counteract ROS generation induced by gp120 does not account for the reported prevention of cell death because ICI 182,780 failed to revert intracellular ROS reduction caused by 17beta-E2 though it was able to revert prevention of cell death. Furthermore, by using 17alpha-E2, the isomer unable to stimulate ER which, however, retains the antioxidant effects, we observed that a pre-treatment with 17alpha-E2 was effective in preventing gp120-induced accumulation of ROS but it failed to affect cell death caused by the viral protein. Collectively, these data demonstrate that neuroprotection afforded by 17beta-E2 is receptor-mediated and ROS scavenging effects may not be implicated.

The effect of inflammatory stimuli on NMDA-related activation of glutamine synthase in human cultured astroglial cells.

Removal of glutamate from the synaptic cleft by astroglial glutamine synthase (GS) is a crucial step in the regulation of glutamate turnover and metabolism, thus participating in endogenous neuroprotective processes occurring within brain tissues. Here we investigated on the effect of inflammatory cytokines on GS activity in astroglial cells undergoing NMDA receptors stimulation. Incubation of human cultured astroglial cells with NMDA (100 microM) enhanced GS expression, an effect driven by the generation of nitric oxide (NO) since l-NAME (500 microM), an inhibitor of NO synthase, reversed this effect. NMDA-related increase of GS activity and glutamine concentration was antagonised by previous incubation of astroglial cells with a mixture of LPS plus gammaIFN, an effect counteracted by dexamethasone, the latter effect being accompanied by inhibition of inducible NO synthase. These results show that LPS plus gammaIFN inhibit elevation of GS activity subsequent to NMDA receptor stimulation in astroglial cells via enhancement of inducible NO synthase, and this may represent the site of interaction between pro-inflammatory and excitotoxic stimuli in the brain.

Estradiol reduces cytochrome c translocation and minimizes hippocampal damage caused by transient global ischemia in rat.

It is well-established that 17beta-estradiol (17beta-E(2)) confers neuroprotection to male and female rats exposed to focal cerebral ischemia, while less is known about the effects of the hormone under conditions of transient global ischemia. Since translocation of cytochrome c from the mitochondria to the cytosol is a critical step in apoptotic cell death after cerebral ischemia, we have investigated whether 17beta-E(2) interferes with such mechanism to exert neuroprotection. Global ischemia, induced in male Wistar rats by 5-min 4 vessel occlusion (4VO), resulted in a significant increase of cytosolic cytochrome c (cyt-c) levels as detected by Western blotting at 6h after reperfusion. 17beta-E(2) (0.2mg/kg, i.p.) given 1h before ischemia minimized cytochrome c translocation and the latter effect was partially reversed by tamoxifen (0.25mg/kg, i.p.). Bilateral cell counting revealed that delayed hippocampal damage typically caused by 4VO was abolished by 17beta-E(2) and this was partially reversed by tamoxifen in the CA3 subregion, but not in CA1/CA2 or CA4. These findings provide the original observation that 17beta-E(2) reduces delayed hippocampal damage caused by 4VO in male rats and blocks cytochrome c translocation during the early stages of neuronal death, thus providing an important mechanism involved in estrogen-mediated neuroprotection.