Animal Models Of Neurological Disorders Research Articles

Comparative study of quercetin and its two glycoside derivatives quercitrin and rutin against methylmercury (MeHg)-induced ROS production in rat brain slices

The hypothesis that methylmercury (MeHg) potently induces formation of reactive oxygen species (ROS) in the brain is supported by observations on the neuroprotective effects of various classes of antioxidants. Flavonoids have been reported to possess divalent metal chelating properties, antioxidant activities and to readily permeate the blood-brain barrier. They can also provide neuroprotection in a wide array of cellular and animal models of neurological diseases. Paradoxically, in vivo administration of quercetin displays unexpected synergistic neurotoxic effect with MeHg. Considering this controversy and the limited data on the interaction of MeHg with other flavonoids, the potential protective effect of quercetin and two of its glycoside analogs (i.e., rutin and quercitrin) against MeHg toxicity were evaluated in rat cortical brain slices. MeHg (100 microM) caused lipid peroxidation and ROS generation. Quercitrin (10 microg/mL) and quercetin (10 microg/mL) protected mitochondria from MeHg (5 microM)-induced changes. In contrast, rutin did not afford a significant protective effect against MeHg (100 microM)-induced lipid peroxidation and ROS production in cortical brain slices. MeHg-generated ROS in cortical slices was dependent upon an increase in intracellular Ca(2+) levels, because the over-production of MeHg-induced H(2)O(2) in mitochondria occurred with a concomitant increase in Ca(2+) transient. Here, we have extended the characterization of mechanisms associated with the neuroprotective effects of quercetin against MeHg-induced toxicity in isolated mitochondria, by performing an array of parallel studies in brain slices. We provide novel data establishing that (1) Ca(2+) plays a central role in MeHg toxicity and (2) in brain slices MeHg induces mitochondrial oxidative stress both via direct interaction with mitochondria (as previously reported in in vitro studies) as well as via mitochondria-independent (or indirect) mechanisms.

Silibinin attenuates cognitive deficits and decreases of dopamine and serotonin induced by repeated methamphetamine treatment

Cognitive deficits are a core feature of patients with methamphetamine (METH) abuse. It has been reported that repeated METH treatment impairs long-term recognition memory in the novel object recognition test (NORT) in mice. Recent studies indicate that silibinin, a flavonoid derived from the herb milk thistle, has potent neuroprotective effects in cell cultures and several animal models of neurological diseases. However, its effect on the cognitive deficit induced by METH remains unclear. In the present study, we attempt to clarify the effect of silibinin on impairments of recognition memory caused by METH in mice. Mice were co-administered silibinin with METH for 7 days and then cognitive function was assessed by NORT after 7-day withdrawal. Tissue levels of dopamine and serotonin as well as their metabolites in the prefrontal cortex and hippocampus were measured 1 day after NORT. Silibinin dose-dependently ameliorated the impairment of recognition memory caused by METH treatment in mice. Silibinin significantly attenuated the decreases in the dopamine content of the prefrontal cortex and serotonin content of the hippocampus caused by METH treatment. We also found a correlation between the recognition values and dopamine and serotonin contents of the prefrontal cortex and hippocampus. The effect of silibinin on cognitive impairment may be associated with an amelioration of decreases in dopamine and serotonin levels in the prefrontal cortex and hippocampus, respectively. These results suggest that silibinin may be useful as a pharmacological tool to investigate the mechanisms of METH-induced cognitive impairments.

A PPARdelta Agonist Reduces Amyloid Burden and Brain Inflammation in a Transgenic Mouse Model of Alzheimers Disease

Agonists of the peroxisome proliferator activated receptor gamma (PPARgamma) have been shown to reduce inflammatory responses in several animal models of neurological diseases and conditions and to reduce amyloid burden in transgenic mice expressing mutant forms of human amyloid precursor protein. However, the effects of activating the related receptor PPARdelta (PPARdelta), which is expressed at higher levels in the brain than PPARgamma, on inflammation and amyloid burden have not been explored. In this study we tested the effects of the selective PPARdelta agonist GW742 in 5xFAD mice which harbor 3 mutations in amyloid precursor protein and 2 mutations in presenilin 1, develop plaques by 5-6 weeks of age, and show robust inflammation and neuronal damage. Oral delivery of GW742 significantly reduced amyloid plaque burden in the subiculum region of 3-month old male and female 5xFAD mice. GW742 also significantly reduced astrocyte activation, suggesting anti-inflammatory effects on glia cells. The changes in plaque burden were accompanied by increased expression of the amyloid degrading enzymes neprilysin and insulin degrading enzyme, while in transfected HEK293 cells, GW742 activated a neprilysin promoter driving luciferase expression. These results suggest that, as found for some PPARgamma agonists, PPARdelta agonists can also reduce amyloid burden likely to be mediated by effects on amyloid clearance.

Systemic Neurotransplantation - A Problem-Oriented Systematic Review

Systemic neurotransplantation (SNT) was introduced in the laboratory in 2000 and currently it is being widely examined in animal models of neurological disorders. The aim of this systematic review was to evaluate the current state of knowledge in the field of experimental SNT and the premise for the introduction of clinical trials. PubMed was searched and 60 articles utilizing an SNT approach were found and subjected to analysis. The time window for cell transplantation was addressed in only two studies, with contradictory results. Immunosuppression was applied in 25% of studies. No study addressed the justification for immunosuppression. Bone marrow was the most frequent source of grafted cells, followed by cord blood and then by cells of embryonic origin. Studies investigating dose-dependency revealed no satisfactory results with transplantation of less than 10(6) cells/animal; the efficient dose most frequently ranged from 10(6)-10(7) cells/animal (mice and rats). The behavioral effects of cell transplantation were assessed in 75% of all studies; significant improvement was achieved in 95% of them. Morphological effect was evaluated in half of the studies; significant positive effect was achieved in 73% of them. Experimental attempts to elucidate the mechanisms mediating cell-dependent effect were not undertaken in half of the studies. In the other half, the most frequent mechanisms were growth factors, neurogenesis and immunomodulation. SNT still seems to be at the very initial stage of development. Many critical factors have not been sufficiently addressed in laboratory studies and they must be clarified before the introduction of clinical trials.

Large Animal Models of Neurological Disorders for Gene Therapy

he development of therapeutic interventions for genetic disorders and diseases that affect the central nervous system (CNS) has proven challenging. There has been significant progress in the development of gene therapy strategies in murine models of human disease, but gene therapy outcomes in these models do not always translate to the human setting. Therefore, large animal models are crucial to the development of diagnostics, treatments, and eventual cures for debilitating neurological disorders. This review focuses on the description of large animal models of neurological diseases such as lysosomal storage diseases, Parkinsons disease, Huntingtons disease, and neuroAIDS. The review also describes the contributions of these models to progress in gene therapy research.

Idazoxan attenuates spinal cord injury by enhanced astrocytic activation and reduced microglial activation in rat experimental autoimmune encephalomyelitis

Idazoxan, an imidazoline 2 receptor (I 2R) ligand, has been shown to protect against brain injury in several animal models of neurological disorders. In the present study we investigated the effect of idazoxan on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. EAE was induced by immunizing Wistar rats with guinea pig spinal cord homogenates emulsified in CFA, followed by daily treatment of idazoxan (0, 0.5 mg/kg, 1.5 mg/kg, 4.5 mg/kg, i.p, bid) for 10 days. The results showed that the treatment of idazoxan (1.5 mg/kg and 4.5 mg/kg) significantly decreased the incidence and alleviated inflammatory cell infiltration and demyelination in spinal cords and cerebral cortex. Furthermore, the protective effect of idazoxan on EAE was associated with the enhanced astrocytic activation and attenuated microglial activation and with the subsequent down-regulated expression of proinflammatory cytokines IL-12p40 and IFN-γ and up-regulated expression of anti-inflammatory cytokines IL-10 and TGF-β 1. Thus, the daily treatment of the I 2R ligand idazoxan for 10 days attenuates EAE pathology by differential modulation of astrocytic and microglial activations, raising a possibility that the I 2R ligand may be a novel strategy for treating EAE.

Simultaneous Manipulation of Multiple Brain Targets by Green Tea Catechins: A Potential Neuroprotective Strategy for Alzheimer and Parkinson Diseases

Current therapeutic approaches for Alzheimer and Parkinson disease (AD and PD, respectively) are merely symptomatic, intended for the treatment of symptoms, but offer only partial benefit, without any disease-modifying activity. Novel promising strategies suggest the use of antiinflammatory drugs, antioxidants, iron-complexing molecules, neurotrophic factor delivery, inhibitors of the amyloid precursor protein (APP)-processing secretases, gamma and beta (that generate the amyloid-beta peptides, Abeta), anti-Abeta aggregation molecules, the interference with lipid cholesterol metabolism and naturally occurring plant flavonoids to potentially reverse the course of the diseases. Human epidemiological and new animal data suggest that tea drinking may decrease the incidence of dementia, AD, and PD. In particular, its main catechin polyphenol constituent (-)-epigallocatechin-3-gallate (EGCG) has been shown to exert neuroprotective/neurorescue activities in a wide array of cellular and animal models of neurological disorders. In the current article, we review the literature on the impact of the multimodal activities of green tea polyphenols and their neuroprotective effect on AD and PD.

Cell Signaling Pathways and Iron Chelation in the Neurorestorative Activity of Green Tea Polyphenols: Special Reference to Epigallocatechin Gallate (EGCG)

Although much progress has been made in understanding the pathogenesis of Alzheimer's disease (AD), the current therapeutic approaches are merely symptomatic, intended for the treatment of cognitive symptoms, such as disturbances in memory and perception. Novel promising strategies suggest the use of anti-inflammatory drugs, antioxidants including natural occurring plant flavonoids, iron-complexing molecules, neurotrophic factor delivery, inhibitors of the amyloid-beta protein precursor processing secretases, gamma and beta, that generate amyloid-beta peptides and the interference with lipid and cholesterol metabolism. Human epidemiological and new animal data suggest that tea drinking may decrease the incidence of dementia, AD and Parkinson's disease. In particular, its main catechin polyphenol constituent (-)-epigallocatechin-3-gallate (EGCG) has been shown to exert neuroprotective/neurorescue activities in a wide array of cellular and animal models of neurological disorders. This review provides a detailed overview on the multimodal activities of green tea polyphenols with emphasis on their iron chelating, neurorescue/neuroregenerative and mitochondrial stabilization action.

Genetic inactivation of adenosine A 2A receptors attenuates acute traumatic brain injury in the mouse cortical impact model

The inactivation of the A 2A receptor (A 2AR) has been shown to neuroprotect against brain injury in several animal models of neurological disorders including stroke and Parkinson's disease. However, despite marked elevation of adenosine level, the role of the A 2A in traumatic brain injury (TBI) remains unclear. In the present study, we investigated the effects of genetic inactivation of A 2ARs in the acute stage. The A 2AR knock-out (KO) mice and their wild-type (WT) littermates were subjected to cortical impact injury by a dropping weight. The control group was only craniotomized without TBI. At 24 h post-TBI, the neurological deficit scores of the KO mice were significantly lower than that of WT littermates. Consistent with the behavioral changes, the brain water contents as well as histological changes and the TUNEL-positive cells of the injured cortex of the KO mice were significantly lower than that of WT littermates. Furthermore, the glutamate level in the cerebral spinal fluid (CSF) of the KO mice was also significantly lower than that of WT littermates. In addition, we found that at 12 h post-TBI the mRNA and protein levels of TNF-α and IL-1β were higher in the KO mice than that in the WT littermates. However, at 24 h post-TBI, the level of TNF-α and IL-1β continually increased in the WT mice but largely declined in the KO mice. These results suggest that the genetic inactivation of A 2AR protects against TBI, which is mainly associated with the suppression of glutamate level.

Targeting Multiple Neurodegenerative Diseases Etiologies with Multimodal-Acting Green Tea Catechins

Green tea is currently considered a source of dietary constituents endowed with biological and pharmacological activities relevant to human health. Human epidemiological and new animal data suggest that the pharmacological benefits of tea drinking may help to protect the brain as we age. Indeed, tea consumption is inversely correlated with the incidence of dementia and Alzheimer’s and Parkinson’s diseases. In particular, its main catechin polyphenol constituent (−)-epigallocatechin-3-gallate has been shown to exert neuroprotective/neurorescue activities in a wide array of cellular and animal models of neurological disorders. The intense efforts dedicated in recent years to shed light on the molecular mechanisms participating in the brain protective action of green tea indicate that in addition to the known antioxidant activity of catechins, the modulation of signal transduction pathways, cell survival/death genes, and mitochondrial function all contribute significantly to the induction of neuron viability. Because of the multietiological character of neurodegenerative disease pathology, these natural compounds are receiving significant attention as therapeutic cytoprotective agents that simultaneously manipulate multiple desired targets in the central nervous system. This article elaborates on the multimodal activities of green tea polyphenols with emphasis on their recently described neurorescue/neuroregenerative and mitochondrial stabilization actions.

Perspectives of in Vivo Magnetic Resonance Imaging of Cell Dynamics in the Brain

MRI is an established diagnostic tool, but it also has great attraction for use in experimental research, particularly in neuroscience and neurology. In vivo imaging of specific cell populations in the brain is particularly attractive for furthering understanding of cell behavior in animal models of neurological disease and injury. Approaches towards this end typically make use of iron oxide nanoparticles as MRI contrast agents. These contrast agents can be taken up by peripheral inflammatory cells, by endogenous CNS cell populations, or by in vitro cell cultures for transplantation experiments. Molecular imaging of functional cell status, using MRI in combination with molecular biology, is a rapidly expanding field with great promise. The present review summarizes the current status of cellular MRI in the brain in the context of ischemia models, and relevant issues and approaches that aim to improve translation of cell therapy strategies into the clinic.

Modulation of Ischemic Brain Injury and Neuroinflammation by Adenosine A2A Receptors

Over the past 5 years, the adenosine A(2A) receptor (A(2A)R) is emerging as an attractive therapeutic target for modulating brain injury in a variety of animal models of neurological disorders including stroke. The evidence we have to date indicates that both adenosine and A(2A) antagonists are neuroprotective in ischaemic brain injury. From drug development perspective, administering A(2A) antagonists in association with inhibitors of adenosine kinase may represent a novel strategy for treating stroke. Despite the well-documented neuroprotection by A(2A)R antagonists, the mechanism by which A(2A)Rs affect brain injury remains largely unknown. In this section, we also summarize the experimental evidence for A(2A)R modulation of glial function as possible contribution to the modulation of brain injury. In vitro and in vivo studies reveal that in response to local neuroinflammation following brain insults, time-dependent, inflammatory signal-mediated induction of the A(2A)R in glial cells (particularly microglial cells) make this cell type particularly sensitive to A(2A)R modulation of brain injury. Furthermore, in contrast to the generally held view that the A(2A)R exerts predominantly anti-inflammatory effects (based upon studies in peripheral organs), the A(2A)R modulation of neuroinflammation may differentially affect the outcome of brain injury, depending on the nature of brain insults. Thus, in association with their ability to reduce brain injury, inactivation of the A(2A)R in most models and activation of A(2A)R in some cases have been shown to attenuate brain inflammation through control of the proliferation and production of proinflammatory cytokines.

The Changing Face of Neural Stem Cell Therapy in Neurologic Diseases

New insights into the biology of neural stem cells (NSCs) have raised expectations for their use in the treatment of neurologic diseases. Originally, NSC transplantation was proposed as a means of replacing cells in central nervous system diseases that result in cell loss. However, recent data regarding their beneficial effects in various animal models of neurologic diseases indicate that transplanted NSCs may also attenuate deleterious inflammation, protect the central nervous system from degeneration, and enhance endogenous recovery processes. Herein, we review recent developments and future prospects of NSC therapy in neurologic diseases.

Mesenchymal Stem Cells as Mediators of Neural Differentiation

Mesenchymal stem cells (MSCs) represent a promising source of material for autologous cell transplantation therapies, in particular, their potential use for the treatment of damaged nervous tissue. Much of the work in this area has focused on the transplantation of MSCs into animal models of neurological disorders, including stroke and spinal cord injury. Although numerous studies have reported significant functional improvements in these systems, the exact mechanism(s) by which MSCs elicit recovery remains largely undefined. While it has been proposed that 'trans'-differentiation and/or cell fusion events underly MSC-mediated neural repair, there is considerable doubt that the low frequency of these phenomena is sufficient to account for the observed levels of recovery. Furthermore, in vitro studies call into question the ability of MSCs to produce authentic neural derivatives. In this review we focus on recent evidence indicating that transplanted MSCs promote endogenous repair of neurologically damaged areas via the release of soluble trophic factors and cytokines. Through the modern analysis of MSC-conditioned media it is becoming possible to gain new insight into the release and interplay of these soluble factors and their neurogenic effects. Ultimately this understanding may lead to the rational design of new therapies for the treatment of neurological and neurodegenerative disorders.

The nose may help the brain: intranasal drug delivery for treating neurological diseases

While enormous progress has been made regarding our understanding of the pathogenic mechanisms of neurological diseases, there are only a small number of effective drugs for treating these illnesses. A key obstacle for developing effective drugs for treating neurological diseases is the blockage of drug entrance into the CNS by the BBB [1]. Less than 2% of all small-molecule drugs, and virtually no large-molecule drugs, can cross the BBB. Therefore, it is of critical significance to search for drug-delivery strategies that can effectively deliver drugs into the CNS. An increasing number of studies on both animals and human subjects have suggested that intranasal drug delivery could be used to deliver both smalland largesized drugs into the CNS by bypassing the BBB. It appears increasingly reasonable to conduct clinical trials to determine if intranasal drug delivery may be used to treat neurological diseases. In the 1970s and 1980s there were multiple studies suggesting that intranasal administration may enable substances to directly enter into the brain by pathways involving the olfactory epithelium and olfactory bulb [2]. In 1995 Thorne et al. reported the first quantitative study indicating that intranasal administration could deliver large-sized molecules into the brain by bypassing the BBB [3]. Intranasal administration of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) led to a significant presence of WGA-HRP in the olfactory bulb of rats, while there was no detectable amount of WGA-HRP in the olfactory bulb after intravenous injection of the same concentration of WGA-HRP. Since 1997, many studies have indicated that intranasal administration can enable large-sized molecules, such as IGF-1, FGF-2, TGF-β1, erythropoietin, IFN-β, HIV-1 Tat, insulin and leptin, to be transported into the CNS at least partially through direct nose-to-brain routes [2,4–9]. A number of studies using animal models of neurological diseases have demonstrated that intranasal delivery of large-sized molecules can produce beneficial effects. For example, intranasal nerve growth factor (NGF) administration can attenuate memory deficits and neurodegeneration in transgenic models of Alzheimer’s disease (AD) [10]; administration of erythropoietin [8] or IGF-I [11] by the intranasal approach can significantly decrease ischemic brain damage; and intranasal delivery of growth factors can also increase neurogenesis in rat brains [9]. We have conducted studies to directly compare the efficacy of intranasal drug delivery with that of intravenous drug delivery in treating brain ischemia. Based on the cell-culture studies showing the protective effects of gallotannin (GT) and nobotanin B – two inhibitors of poly(ADP-ribose) glycohydrolase – against oxidative cell injury [12], we found that intranasal delivery of GT is much more effective than intravenous injection of GT in decreasing ischemic brain injury [13]. On the basis of the in vitro findings that nicotinamide adenine dinucleotide (NAD+) treatment can prevent oxidative stress-induced cell death [14], we also found that intranasal NAD+ administration can produce up to 90% decreases in infarct formation when given 2 h after ischemia, which is the most profound protection ever reported from drugs that have been administered hours after ischemic onset [15,16].

Stem cell‐based cell therapy for Huntington disease: A review

Huntington disease (HD) is a devastating neurodegenerative disorder and no proven medical therapy is currently available to mitigate its clinical manifestations. Although fetal neural transplantation has been tried in both preclinical and clinical investigations, the efficacy is not satisfactory. With the recent explosive progress of stem cell biology, application of stem cell-based therapy in HD is an exciting prospect. Three kinds of stem cells, embryonic stem cells, bone marrow mesenchymal stem cells and neural stem cells, have previously been utilized in cell therapy in animal models of neurological disorders. However, neural stem cells were preferably used by investigators in experimental HD studies, since they have a clear capacity to become neurons or glial cells after intracerebral or intravenous transplantation, and they induce functional recovery. In this review, we summarize the current state of cell therapy utilizing stem cells in experimental HD animal models, and discuss the future considerations for developing new therapeutic strategies using neural stem cells.

Multilineage Potential of Stable Human Mesenchymal Stem Cell Line Derived from Fetal Marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and β-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.

Improvement by minocycline of methamphetamine-induced impairment of recognition memory in mice

Cognitive deficits are a core feature of patients with schizophrenia and methamphetamine (METH) psychosis. We have recently found that repeated METH treatment (1 mg/kg, s.c.) in mice, which induces behavioral sensitization, impairs long-term recognition memory in a novel object recognition test (NORT) and that the impairment is ameliorated by clozapine, but not haloperidol. Recent studies indicate that minocycline, a second-generation tetracycline, has potent neuroprotective effects in various animal models of neurological diseases. In the present study, we investigated the effect of minocycline on learning and memory in the NORT and behavioral sensitization in mice that had been administered METH for 7 days. When minocycline (20-40 mg/kg) was administered intraperitoneally once a day for seven consecutive days to mice that had previously been treated with METH for 7 days, it ameliorated the METH-induced impairment of recognition memory in a dose-dependent manner, although the same treatment with minocycline had no effect on behavioral sensitization to METH. The administration of minocycline, together with METH, inhibited the development of METH-induced behavioral sensitization. The improvement in memory caused by minocycline was associated with an amelioration of the novelty-induced activation of extracellular signal-regulated kinase 1/2 in the prefrontal cortex of METH-treated mice. These results suggest that minocycline is useful for the treatment of cognitive deficits in patients with METH psychosis or schizophrenia.

Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development

Large-scale proteomic analysis of the mammalian brain has been successfully performed with mass spectrometry techniques, such as Multidimensional Protein Identification Technology (MudPIT), to identify hundreds to thousands of proteins. Strategies to efficiently quantify protein expression levels in the brain in a large-scale fashion, however, are lacking. Here, we demonstrate a novel quantification strategy for brain proteomics called SILAM (Stable Isotope Labeling in Mammals). We utilized a (15)N metabolically labeled rat brain as an internal standard to perform quantitative MudPIT analysis on the synaptosomal fraction of the cerebellum during post-natal development. We quantified the protein expression level of 1138 proteins in four developmental time points, and 196 protein alterations were determined to be statistically significant. Over 50% of the developmental changes observed have been previously reported using other protein quantification techniques, and we also identified proteins as potential novel regulators of neurodevelopment. We report the first large-scale proteomic analysis of synaptic development in the cerebellum, and we demonstrate a useful quantitative strategy for studying animal models of neurological disease.

Neuroprotective effects of anthocyanins and their in vivo metabolites in SH-SY5Y cells

Recent in vivo studies have highlighted an important role for the neuroprotective actions of dietary anthocyanins. However, one consistent result of these studies is that the systemic bioavailability of anthocyanins, including cyanidin 3-O-glucopyranoside (Cy-3G), is very poor. Cy-3G has been demonstrated to be highly instable at physiological pH, so its in vivo metabolites, such as the aglycon cyanidin (Cy) and protocatechuic acid (PA), may be responsible for both the antioxidant activitiy and the neuroprotective effects observed in vivo. Therefore, we investigated the protective effects of Cy-3G, Cy and PA against H(2)O(2)-induced oxidative stress in a human neuronal cell line (SH-SY5Y). We determined their ability to counteract reactive oxygen species (ROS) formation and to inhibit apoptosis in terms of mitochondrial functioning loss and DNA fragmentation induced by H(2)O(2). We demonstrated that pretreatment of SH-SY5Y cells with Cy-3G, Cy and PA inhibits H(2)O(2)-induced ROS formation at different cellular levels: Cy-3G at membrane level, PA at cytosolic level and Cy at both membrane and cytosolic levels. In addition, Cy showed a higher antioxidant activity at membrane and cytosolic level than Cy-3G and PA, respectively. Interestingly, both Cy and PA, but not Cy-3G, could inhibit H(2)O(2)-induced apoptotic events, such as mitochondrial functioning loss and DNA fragmentation. These results suggest that Cy and PA may be considered as neuroprotective molecules and may play an important role in brain health promotion. These in vitro findings should encourage further research in animal models of neurological diseases to explore the potential neuroprotective effects of compounds generated during in vivo metabolism of anthocyanins.