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Cannabinoids derived from Cannabis sativa demonstrate neuroprotective properties in various cellular and animal models. Mitochondrial impairment and consecutive oxidative stress appear to be major molecular mechanisms of neurodegeneration. Therefore we studied some major cannabinoids, i.e. delta-9-tetrahydrocannabinolic acid (THCA), delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in mice mesencephalic cultures for their protective capacities against 1-methyl-4-phenyl pyridinium (MPP(+)) toxicity. MPP(+) is an established model compound in the research of parkinsonism that acts as a complex I inhibitor of the mitochondrial respiratory chain, resulting in excessive radical formation and cell degeneration. MPP(+) (10[mu]M) was administered for 48h at the 9th DIV with or without concomitant cannabinoid treatment at concentrations ranging from 0.01 to 10[mu]M. All cannabinoids exhibited in vitro antioxidative action ranging from 669±11.1 (THC), 16±3.2 (THCA) to 356±29.5 (CBD) [mu]g Trolox (a vitamin E derivative)/mg substance in the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay. Cannabinoids were without effect on the morphology of dopaminergic cells stained by tyrosine hydroxylase (TH) immunoreaction. THC caused a dose-dependent increase of cell count up to 17.3% at 10[mu]M, whereas CBD only had an effect at highest concentrations (decrease of cell count by 10.1-20% at concentrations of 0.01-10[mu]M). It influenced the viability of the TH immunoreactive neurons significantly, whereas THCA exerts no influence on dopaminergic cell count. Exposure of cultures to 10[mu]M of MPP(+) for 48h significantly decreased the number of TH immunoreactive neurons by 44.7%, and shrunken cell bodies and reduced neurite lengths could be observed. Concomitant treatment of cultures with cannabinoids rescued dopaminergic cells. Compared to MPP(+) treated cultures, THC counteracted toxic effects in a dose-dependent manner. THCA and CBD treatment at a concentration of 10[mu]M lead to significantly increased cell counts to 123% and 117%, respectively. Even though no significant preservation or recovery of neurite outgrowth to control values could be observed, our data show that cannabinoids THC and THCA protect dopaminergic neurons against MPP(+) induced cell death. [ABSTRACT FROM AUTHOR]

A time span of 60 years covers the detection of catecholamines in the brain, their function in movement and correlation to Parkinson's disease (PD). The clinical findings that orally given L-DOPA can alleviate or even prevent akinesia gave great hope for the treatment of PD. Attention focused on the role of tyrosine hydroxylase (TH) as the rate-limiting enzyme in the formation of catecholamines. It became evident that the enzyme driven formation is lowered in PD. Such results could only be obtained from studying human brain samples demonstrating the necessity for human brain banks. Originally, a TH enzyme deficiency was suspected in PD. Studies were conducted on the enzyme properties: its induction and turnover, the complex regulation starting with cofactor requirements as tetrahydrobiopterin and ferrous iron, and the necessity for phosphorylation for activity as well as inhibition by toxins or regulatory feedback inhibition by catecholamines. In the course of time, it became evident that neurodegeneration and cell death of dopaminergic neurons is the actual pathological process and the decrease of TH a cophenomenon. Nevertheless, TH immunochemistry has ever since been a valuable tool to study neuronal pathways, neurodegeneration in various animal models of neurotoxicity and cell cultures, which have been used as well to test potential neuroprotective strategies., (© 2022. The Author(s).)

Iron plays a crucial role in many physiological processes of the human body, but iron is continuously deposited in the brain as we age. Early studies found iron overload is directly proportional to cognitive decline in Alzheimer's disease (AD). Amyloid precursor protein (APP) and tau protein, both of which are related to the AD pathogenesis, are associated with brain iron metabolism. A variety of iron metabolism-related proteins have been found to be abnormally expressed in the brains of AD patients and mouse models, resulting in iron deposition and promoting AD progression. Amyloid β (Aβ) and hyperphosphorylated tau, two pathological hallmarks of AD, can also promote iron deposition in the brain, forming a vicious cycle of AD development-iron deposition. Iron deposition and the subsequent ferroptosis has been found to be a potential mechanism underlying neuronal loss in many neurodegenerative diseases. Iron chelators, antioxidants and hepcidin were found useful for treating AD, which represents an important direction for AD treatment research and drug development in the future. The review explored the deep connection between iron dysregulation and AD pathogenesis, discussed the potential of new hypothesis related to iron dyshomeostasis and ferroptosis, and summarized the therapeutics capable of targeting iron, with the expectation to draw more attention of iron dysregulation and corresponding drug development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Wang, Shen, Li, Rausch and Huang.)

Rotenone ([2R-(2α,6aα,12aα)]-1,2,12,12a-tetrahydro-8,9-dimethoxy-2-(1-methylethenyl)-[1]benzopyran[3,4-b]furo [2,3-h][1]benzopyran-6(6aH)-one) is a naturally occurring compound derived from the roots and stems of Derris, Tephrosia, Lonchocarpus and Mundulea plant species. Since its discovery at the end of the 19th century, rotenone has been widely used as a pesticide for controlling insects, ticks and lice, and as a piscicide for management of nuisance fish in lakes and reservoirs. In 2000, Betarbet et al. reproduced most of the behavioural, biochemical and pathological features of Parkinson's disease (PD) in rotenone-treated rats. Since that time, rotenone has received much attention as it would be one of the environmental neurotoxins implicated in etiopathogenesis of PD. Moreover, it represents a common experimental model to investigate the underlying mechanisms leading to PD and evaluate the new potential therapies for the disease. In the current general review, we aimed to address recent advances in the hazards of the environmental applications of rotenone and discuss the updates on the rotenone model of PD and whether it is implicated in the etiopathogenesis of the disease.

Introduction: Exposure to acrylamide is increasing worldwide as a result of its heavy use in industry and formation in carbohydrate-rich food cooked at high temperature. Despite its neurotoxicity, no studies have shown its toxic effects on dopaminergic neurons yet. Therefore, the current study was carried out to show whether acrylamide adversely affects primary cultured dopaminergic neurons., Material and Methods: Acrylamide (0.001, 0.01, 0.1, 1, 2 mM) was added to two different groups of primary mesencephalic cell cultures on the 9th day in vitro for 24 and 48 h, respectively. Moreover, a group of cultures was treated with lower concentrations of acrylamide (0.01, 0.05, 0.1, 0.5 mM) on the 6th day in vitro for 5 consecutive days to investigate its long-term effects on dopaminergic neurons. Following each treatment, culture media were obtained for measuring lactate dehydrogenase, and cultured cells were stained immunocytochemically against tyrosine hydroxylase and neuronal nuclear antigens., Results: Treatment of cultures with acrylamide for 48 h significantly reduced the number of dopaminergic neurons, adversely altered the morphology of the surviving neurons and increased levels of lactate dehydrogenase in the culture media. Similar treatment of cultures with acrylamide also resulted in lower numbers of total neuronal cells as shown by a reduced expression of the neuronal nuclear antigen. Prolonged treatment of cultures with lower concentrations of acrylamide slightly reduced the survival of dopaminergic neurons but increased the release of lactate dehydrogenase into the culture media as well., Conclusions: The current study shows, for the first time, neurotoxicity of acrylamide on dopaminergic neurons in the primary mesencephalic cell culture.

Vascular dementia (VaD) is the second most common type of dementia after Alzheimer's disease (AD), characterized by progressive cognitive impairment, memory loss, and thinking or speech problems. VaD is usually caused by cerebrovascular disease, during which, cerebrovascular endothelial cells (CECs) are vulnerable. CEC dysfunction occurs before the onset of VaD and can eventually lead to dysregulation of cerebral blood flow and blood-brain barrier damage, followed by the activation of glia and inflammatory environment in the brain. White matter, neuronal axons, and synapses are compromised in this process, leading to cognitive impairment. The present review summarizes the mechanisms underlying CEC impairment during hypoperfusion and pathological role of CECs in VaD. Through the comprehensive examination and summarization, endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway, Ras homolog gene family member A (RhoA) signaling pathway, and CEC-derived caveolin-1 (CAV-1) are proposed to serve as targets of new drugs for the treatment of VaD.

Domoic acid, an excitatory neurotoxin produced by certain algae, reaches the food chain through accumulation in some sea organisms. To investigate its long-term neurotoxicity on dopaminergic neurons, prepared primary mesencephalic cell cultures were exposed to different concentrations of domoic acid (0.1, 1, 10, 100 μM) on the 8th day in vitro (DIV) for 4 days. On the 12th DIV, culture media were collected for measurement of lactate dehydrogenase and cultured cells were subjected to immunohistochemistry against tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H 2 DCFDA, JC-1 and Hoechst 33342 dyes. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. Domoic acid significantly decreased the number of dopaminergic neurons and adversely affected their morphology, and slightly reduced the expression of neuronal nuclear antigen and glial fibrillary acidic protein. Co-treatment of cultures with domoic acid and the AMPA/KA or NMDA receptor antagonists NBQX and MK-801 rescued significant number of dopaminergic neurons. Domoic acid significantly decreased red:green fluorescence ratio of JC-1 and did not affect production of reactive oxygen species and apoptotic cell death. In conclusions, the present study reveals that long-term treatment of primary mesencephalic cell culture with domoic acid significantly destroyed dopaminergic neurons. This effect appears to be attributed to activation of AMPA/KA and NMDA receptors and mitochondrial damage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nuclear receptor-related factor 1 (Nurr1) has a crucial role in the development and maturation of mesencephalic dopamine (DA) neurons and also plays a protective role in maintenance of DA neurons by inhibiting the activation of microglia and astrocyte. Moreover, the mutations in Nurr1 gene are associated with familial Parkinson's disease (PD), suggested that Nurr1 modulation is a potential therapeutic target for PD. This study examines the therapeutic effects of transplantation of Nurr1 gene-modified bone marrow mesenchymal stem cells (MSCs) on 6-hydroxydopamine (6-OHDA)-induced PD rat models. MSCs were transduced with lentivirus expressing Nurr1 gene and then intrastriatally transplanted into PD rats. Our results showed that Nurr1 gene-modified MSCs overexpress and secrete Nurr1 protein in vitro and also survive and migrate in the brain. Four weeks after transplantation Nurr1 gene-modified MSCs dramatically ameliorated the abnormal behavior of PD rats and increased the numbers of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum, inhibited the activation of glial cells, and reduced the expression of inflammatory factors in the SN. Taken together, these findings suggest that intrastriatal transplantation of lentiviral vector mediated Nurr1 gene-modified MSCs has notable therapeutic effect for PD rats., Competing Interests: None.

Introduction: Domoic acid is a potent marine neurotoxin produced by certain species of the diatom genus Pseudonitzschia. To our knowledge, there are no studies that have investigated neurotoxic effects of domoic acid on dopaminergic neurons. Accordingly, the present study was carried out to investigate the potential neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture., Material and Methods: Cultures prepared from embryonic mouse mesencephala (total of 250 embryos) were treated with different concentrations of domoic acid (0.1, 1, 10, 100 µM) on the 10th DIV for 48 h. On the 12th DIV, culture media were used for measurement of lactate dehydrogenase and cultured cells were subjected to immunostaining for tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and DAPI stains. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated., Results: Domoic acid significantly decreased the number of dopaminergic neurons, decreased the expression of neuronal nuclear antigen and slightly affected astrocyte populations, and increased the release of lactate dehydrogenase into the culture media. AMPA/KA receptor antagonist NBQX but not NMDA receptor antagonist MK-801 significantly inhibited the neurotoxic effect of domoic acid on dopaminergic neurons. H₂DCFDA, JC-1 and DAPI fluorescence staining, respectively, revealed that DomA slightly raised ROS production, and significantly decreased mitochondrial membrane potential and increased apoptotic cell death of cultured cells., Conclusion: The current study presents for the first time the neurotoxic effects of domoic acid on dopaminergic neurons and this effect appears to be attributed to activation of AMPA/KA receptors on dopaminergic neurons.

In the course of our search for antitumour constituents from the traditional Mongolian medicinal herb Clematis aethusifolia Turcz., 11 flavonoids were isolated for the first time from the dried aerial parts of the plant by flash C 18 column chromatography, Sephadex LH-20 and reversed phase preparative HPLC. The planar structures of these flavonoids were established based on 1D and 2D NMR and high-resolution mass spectrometry. Compounds 1, 2, 4 and 5 showed moderate cytotoxicity against a panel of five human solid tumour cell lines, including A-375, a human melanoma cell line; SK-OV-3, a human ovarian cancer cell line; A549, a human lung cancer cell line; HCT-15, a human colorectal adenocarcinoma cell line; and SH-SY5Y, a human neuroblastoma cell line (with IC 50 values of 20-70 μM). The obtained cytotoxic apigenin and its derivatives may be useful as standard compounds for the quality control of the crude drug and its preparations.

Background: Generation of nascent functional neurons from neural stem cells in the adult brain has recently become largely accepted by the neuroscience community. In adult mammals including humans, the process of neurogenesis has been well documented in two brain regions; the subventricular zone of the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus., Method: Some evidence has indicated neurogenesis in other regions of the adult mammalian brain such as the neocortex, cerebellum, striatum, amygdala and hypothalamus. These discoveries question a long standing dogma on nervous system regeneration and provide medical science with potential new strategies to harness the process of neurogenesis for treating neurological disabilities and neurodegenerative diseases., Conclusion: In this current review, we address the most recent advances on the role of neurogenesis in the adult brain and therapeutic potential in the two most common neurodegenerative disorders, Parkinson's and Alzheimer's diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Oxidative stress plays a pivotal role in the progressive neurodegeneration in Parkinson's disease (PD) which is responsible for disabling motor abnormalities in more than 6.5 million people worldwide. Polysaccharides are the main active constituents from Ganoderma lucidum which is characterized with anti-oxidant, antitumor and immunostimulant properties. In the present study, primary dopaminergic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effects and the potential mechanisms of Ganoderma lucidum polysaccharides (GLP) on the degeneration of dopaminergic neurons induced by the neurotoxins methyl-4-phenylpyridine (MPP(+)) and rotenone. Results revealed that GLP can protect dopamine neurons against MPP(+) and rotenone at the concentrations of 100, 50 and 25 μg/ml in primary mesencephalic cultures in a dose-dependent manner. Interestingly, either with or without neurotoxin treatment, GLP treatment elevated the survival of THir neurons, and increased the length of neurites of dopaminergic neurons. The Trolox equivalent anti-oxidant capacity (TEAC) of GLP was determined to be 199.53 μmol Trolox/g extract, and the decrease of mitochondrial complex I activity induced by MPP(+) and rotenone was elevated by GLP treatment (100, 50, 25 and 12.5 μg/ml) in a dose dependent manner. Furthermore, GLP dramatically decreased the relative number of apoptotic cells and increased the declining mitochondrial membrane potential (ΔΨm) induced by MPP(+) and rotenone in a dose-dependent manner. In addition, GLP treatment reduced the ROS formation induced by MPP(+) and rotenone at the concentrations of 100, 50 and 25 μg/ml in a dose-dependent manner. Our study indicates that GLP possesses neuroprotective properties against MPP(+) and rotenone neurotoxicity through suppressing oxidative stress in primary mesencephalic dopaminergic cell culture owning to its antioxidant activities.

Ginsenosides are the main active constituents of Panax ginseng. Ginsenoside Re is one of the major ginsenosides; whereas hydrolysis products such as Rd appear to have higher biological activity though are present in smaller amounts. Ginsenosides, from their early use in folk medicine to modern studies, appear to exert beneficial actions against aging and even neurodegenerative disorders. Parkinson's disease is a progressive neurodegenerative movement disorder characterized by a profound loss of midbrain dopamine neurons in the substantia nigra pars compacta. Carbon tetrachloride (CCl4) exerts neurotoxic effects when present as an environmental pollutant. As a model compound it was used here to study the impact on primary nigrostriatal dopaminergic nerve cells and to investigate the neuroprotective potential of ginsenosides Rd and Re against this organic solvent. CCl4 (2.5 mM on day 12 in vitro for 48 h) significantly decreased the number of tyrosine hydroxylase (TH+) cells by 51% compared with untreated control cultures, reduced their neuritic lengths, and led to truncated degenerations of cell morphology. Ginsenosides Rd and Re (10 µM) strongly reduced cell loss and degeneration and significantly protected process lengths and numbers of neurites of TH+ cells. The anti-oxidative and anti-inflammatory potential of the cellular supernatant was lowered by CCl4 exposure. Inclusion of ginsenosides inhibited both oxidative stress and inflammation. Therefore the neuroprotective effects of ginsenosides at least partially depend on lowering oxidative stress and anti-inflammation.

Background: Dopamine agonists are utilized clinically as an initial treatment in younger Parkinson's disease patients to delay the side effects associated with commencement of levodopa medication. These agonists also serveas adjunctive therapeutics with levodopa to lower the incidence of adverse motor symptoms in advanced stages of the disease., Objectives: To compare the neuroprotective effects of the dopamine agonists pergolide and pramipexole on ferrous sulfate-induced neurotoxicity in dopaminergic neurons from primary mesencephalic cell culture., Methods: Pergolide (0.001-1 μM) and pramipexole (0.01-200 μM) were administered to 8 day primary murine mesencephalic cultures for 24 h. in the presence or absence of desferal, sulpiride or cycloheximide. Ferrous sulfate (450 μM) was then added for 24 hrs. Lactate dehydrogenase was assayed in the supernatant, glutathione concentrations measured in cell lysates and fixed cells were stained for tyrosine hydroxylase., Results: Ferrous sulphate induced neurotoxity in cultures (p<0.0001) was abolished in the presence of the iron chelator desferal (p<0.008). Both pergolide (p<0.0001) and pramipexole (p<0.0001) significantly protected dopaminergic neurons against ferrous sulfate induced neurotoxicity and pramipexole helped preserve neurite morphology. Pramipexole treatment significantly reduced lactate dehydrogenase release (p<0.0001) as a measure of cellular injury. The dopamine receptor antagonist sulpiride (p<0.0001) and the protein synthesis inhibitor cycloheximide (p<0.0001) reduced the neuroprotective effects of pergolide indicating the involvement receptor stimulation and de novo protein synthesis in pergolide-mediated neuroprotection. Pramipexole also significantly reversed the decrease in cellular glutathione concentrations induced by ferrous sulfate (p<0.001)., Conclusion: Both pergolide and pramipexole protect dopaminergic neurons against the neurotoxicity of ferrous sulfate. Pergolide specifically protects dopaminergic neurons through activation of dopamine receptors and de novo protein synthesis whereas pramipexole shows an overall effect through an antioxidant mechanism.

Macroautophagy is a highly regulated intracellular process that, under certain circumstances, delivers cytoplasmic components to the lysosomes for degradation. It consists of several sequential steps including initiation and nucleation, double membrane formation and elongation, formation and maturation of autophagosomes and finally autophagosomes/lysosomes fusion and degradation of intra-autophagosomal contents by lysosomal enzymes. After decades of considering autophagy as a cell death pathway, it has recently been shown to have a survival function through clearing of protein aggregates and damaged cytoplasmic organelles in response to a variety of stress conditions. Most recently, there is increasing evidence from literature revealing that autophagy induction may combat neurodegeneration. In the light of this, our current review tried to address the recent advances in the role of induced autophagy in neuroprotection with a particular focus on its contribution in the most common neurodegenerative disorders like Alzheimer's disease, Parkinson's disease and Huntington's disease.

In the present study, primary mesencephalic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effect of cabergoline, an ergoline D2 receptor agonist, against the pesticide and neurotoxin rotenone relevant to Parkinson disease (PD). Treatment of cultures with cabergoline alone significantly increased the number of tyrosine hydroxylase immunoreactive (THir) neurons and reduced the release of lactate dehydrogenase (LDH) into the culture medium compared to untreated controls. Against rotenone toxicity, cabergoline significantly rescued degenerating THir neurons, reduced the release of LDH into the culture medium and improved the morphology of surviving THir neurons. The neuroprotective effects afforded by cabergoline were independent of dopaminergic stimulation as blocking of dopamine receptors by the dopamine receptor antagonist sulpiride did not prevent them. Furthermore, rotenone-induced formation of reactive oxygen species (ROS) was significantly reduced by cabergoline. Although cabergoline increased the glutathione (GSH) content in the culture, the protective effect for dopaminergic neurons seemed not to be predominantly mediated by increasing GSH, as depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), a GSH biosynthesis inhibitor, did not prevent cabergoline-mediated neuroprotection of THir neurons in rotenone-treated cultures. Moreover, cabergoline significantly increased the ATP/protein ratio in primary mesencephalic cell cultures when added alone or prior to rotenone treatment. These results indicate a neuroprotective effect of cabergoline for dopaminergic neurons against rotenone toxicity. This effect was independent of dopamine receptor stimulation and was at least partially mediated by reducing ROS production and increasing the ATP/protein ratio.

Introduction: Parkinson's disease is the most common movement disorder, characterized by a progressive and extensive loss of dopaminergic neurons in the substantia nigra pars compacta and their terminals in the striatum. So far, only symptomatic treatment is available, and no cure or disease-modifying drugs exist. The present study was designed to investigate the neuroprotective effect of rapamycin, an autophagy inducer, on dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture., Material and Methods: Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephala (OFI/SPF, Vienna, Austria) at gestation day 14. Four sets of cultures were treated as follows: one was run as an untreated control, a second one was treated with 20 nM rotenone on the 10th day in vitro (DIV) for 48 h, a third one was co-treated with 20 nM rotenone and rapamycin (1, 10, 100, 1000 nM) on the 10th DIV for 48 h, and a fourth one was treated with rapamycin alone (1, 10, 100, 1000 nM) on the 10th DIV for 48 h. On the 12th DIV, cultures were subjected to immunohistochemistry against tyrosine hydroxylase and to fluorescence staining using LysoTracker Deep Red, JC-1 and DAPI stains., Results: Exposure of such cultures to 20 nM rotenone on the 10th DIV for 48 h reduced the number of dopaminergic neurons by 41% and increased the release of lactate dehydrogenase (LDH) by 178% above untreated controls. Rapamycin (1, 10, 100, 1000 nM) added together with rotenone from the 10th to 12th DIV spared dopaminergic neurons by 17% and reduced the release of LDH by 64% at the concentration of 100 nM compared to rotenone-treated cultures. Activation of an autophagic process by rapamycin was demonstrated by LysoTracker Deep Red fluorescent dye, as indicated by a shift to increased red fluorescence. Rapamycin also significantly elevated the mitochondrial membrane potential (Δψm), as shown by an increase of the red:green fluorescence ratio of JC-1. Increased apoptotic cell death due to rotenone was lowered by rapamycin, as shown by the blue-fluorescent DAPI nucleic acid stain., Conclusions: Our study indicates for the first time that rapamycin, known as an autophagy inducer, protected dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

Objective: To investigate potential mechanisms mediating the neuroprotective effect of thymoquinone (TQ) on dopaminergic neurons., Methods: This study was conducted in the Chemistry and Biochemistry Institute, University of Veterinary Medicine, Vienna, Austria between June and August 2013. Primary cultures were prepared from embryonic mouse mesencephala (OFI/SPF) at gestation day 14. Four sets of cultures were kept untreated, treated with TQ on the eighth day in vitro (DIV) for 4 days, treated with 1-methyl-4-phenylpyridinium (MPP+) on the tenth DIV for 48 hours and co-treated with thymoquinone and MPP+. On the twelfth DIV, cultures were subjected to immunohistochemistry against tyrosine hydroxylase and fluorescent staining using LysoTracker Deep Red, 5,5`,6,6`-tetrachloro-1,1`,3,3`-tetraethylbenzimidazolylcarbocyanine (JC-1) and 4`,6-diamidino-2-phenylindole stains., Results: The MPP+ decreased the number of dopaminergic neurons by 40%, and increased the release of lactate dehydrogenase (LDH) into the culture medium. The TQ significantly rescued dopaminergic neurons and decreased the release of LDH at the concentrations of 0.1 and 1 uM. The TQ significantly shifted the red fluorescent intensity of the LysoTracker Deep Red, increased the mitochondrial membrane potential as it increased the red:green florescent ratio of JC-1, and decreased MPP+-induced apoptotic cell death., Conclusion: The TQ protects dopaminergic neurons in primary mesencephalic culture by enhancing lysosomal degradation that clears damaged mitochondria and inhibits mitochondria-mediated apoptotic cell death.

Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [(3)H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 nM (THCA) to 14711 nM (CBDV), whereas Ki values to CB2 range from 8.5 nM (THC) to 574.2 nM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are not linked to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one. [Corrected], (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Dopamine agonists are suggested to be more efficacious in treating Parkinson's disease (PD) as they have neuroprotective properties in addition to their receptor-related actions., Aim of the Study: The present study was designed to investigate the neuroprotective effects of rotigotine, a D3/D2/D1 dopamine receptor agonist, against the two powerful complex I inhibitors, 1-methyl-4-phenylpyridinium (MPP+) and rotenone, in primary mesencephalic cell culture relevant to PD., Material and Methods: Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephala at gestation day 14. Three sets of cultures were treated with rotigotine alone, rotigotine and MPP⁺, and rotigotine and rotenone to investigate the effect of rotigotine on the survival of dopaminergic neurons against age-, MPP⁺- and rotenone-induced cell death. At the end of each treatment, cultures were fixed and stained immunohistochemically against tyrosine hydroxylase (TH). The effect of rotigotine against rotenone-induced reactive oxygen species (ROS) production was measured using CH-H2DCFDA fluorescence dye., Results: Rotigotine alone did not influence the survival of tyrosine hydroxylase immunoreactive (THir) neurons except at 10 µM, it significantly decreased the number of THir neurons by 40% compared to untreated controls. Treatment of cultures with 0.01 µM rotigotine rescued 10% of THir neurons against MPP⁺-induced cell death. Rotigotine was also found to significantly rescue 20% of THir neurons at 0.01 µM of rotenone-treated cultures. Using of CH-H2DCFDA fluorescence dye, it was found that rotigotine significantly attenuated ROS production compared to rotenone-treated cultures., Conclusions: Rotigotine provides minor protection against MPP⁺ and rescues a significant number of THir neurons against rotenone in primary mesencephalic cell cultures relevant to PD.

The present study aims to investigate the protective effects of thymoquinone, the major active ingredient of Nigella sativa seeds, against lead-induced brain damage in Sprague-Dawley rats. In which, 40 rats were divided into four groups (10 rats each). The first group served as control. The second, third and fourth groups received lead acetate, lead acetate and thymoquinone, and thymoquinone only, respectively, for one month. Lead acetate was given in drinking water at a concentration of 0.5 g/l (500 ppm). Thymoquinone was given daily at a dose of 20mg/kg b.w. in corn oil by gastric tube. Control and thymoquinone-treated rats showed normal brain histology. Treatment of rats with lead acetate was shown to produce degeneration of endothelial lining of brain blood vessels with peri-vascular cuffing of mononuclear cells consistent to lymphocytes, congestion of choroid plexus blood vessels, ischemic brain infarction, chromatolysis and neuronal degeneration, microglial reaction and neuronophagia, degeneration of hippocampal and cerebellar neurons, and axonal demyelination. On the other hand, co-administration of thymoquinone with lead acetate markedly decreased the incidence of lead acetate-induced pathological lesions. Thus the current study shed some light on the beneficial effects of thymoquinone against neurotoxic effects of lead in rats., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Aim: Nitric oxide (NO) plays a crucial role in vascular tone regulation and is involved in pathogenesis of periodontitis. In this cross-sectional study, we investigated the serum and saliva levels of NO metabolites in periodontal disease and their relationship with serum C-reactive protein (CRP) levels, lipids metabolism and periodontal disease severity., Material and Methods: Serum and saliva were collected from non-smoking patients with generalized severe periodontitis (n = 89) and healthy controls (n = 56). Serum and salivary levels of NO metabolites, serum levels of high density lipoproteins (HDL), low density lipoproteins (LDL), triglycerides, cholesterol and CRP were measured. Data were analysed in whole population and in different gender groups., Results: Periodontitis patients exhibited significantly lower serum and saliva levels of NO metabolites and significantly higher LDL, cholesterol and CRP levels than control group. Similar findings were observed within male but not within female population. Serum NO metabolites levels exhibited significant negative correlation with CRP in whole population and in male population. Significant positive correlation of serum NO metabolite levels with HDL levels was observed in whole population., Conclusion: NO production is reduced in periodontitis, especially in male population. Gender might be an important factor in assessing risk of cardiovascular disease in periodontitis., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Resveratrol interacts with the complex III of the respiratory chain, is a radical scavenger and also suppressor of radical formation in the mitochondria. It reduces the intracellular calcium levels in pre- and postsynaptic neurons and also may inhibit the pro-apoptotic factors in glutamate overflow that occurs, e.g. in excitotoxicity. In cell cultures, glutamate overflow leads to formation of free radicals and results in apoptosis. This increase of radical concentration is enhanced by influx of cations like iron or copper ions into the cell. In present study, the beneficial action of resveratrol was investigated in glutamate-affected dissociated cultures of mice mesencephalic primary cultures. On the 10th day in vitro, 5 mM of glutamate was administered for 15 min and the cultures were further maintained in medium containing 0, 0.01, 0.1 or 1 μM of resveratrol. Resveratrol reduced glutamate-induced damages. The number of dopaminergic neurons was increased and their morphology ameliorated when resveratrol followed glutamate treatment. A significant reduction of glutamate-induced radical formation in cultures treated with resveratrol corresponded with a considerable high antioxidative potential of this stilbene determined using the DPPH assay. In addition, ICP-OES was set up to measure the tissues' copper and iron contents in organotypic cortical cultures of glutamate treated (0 or 30 μM) slices and those in which resveratrol (0, 0.01, 0.1 or 1 μM) was co-administered. Levels of copper were dose-dependently increased, and also the concentration of iron was higher in resveratrol-treated organotypic cultures. The hypothesis that resveratrol has beneficial actions against glutamate damages was verified.

Objectives: Melatonin is a hormone, which is involved in the control of the circadian rhythm, but also acts as an antioxidant and immune modulator. Previous studies reported decreased salivary and serum melatonin levels in periodontitis. This prospective cohort trial assessed the effect of non-surgical periodontal therapy on melatonin levels., Methods: Salivary and serum samples of 60 participants (30 patients suffering from a severe generalized form of periodontitis, 30 healthy controls) were collected at baseline and 19 samples of periodontitis patients after treatment. Salivary and serum melatonin levels were determined by a commercially available ELISA kit and serum C-reactive protein (CRP) by a routine laboratory test., Results: At baseline, periodontitis patients showed significantly increased serum CRP values and significantly decreased salivary melatonin levels compared to the control group. Clinical periodontal parameters significantly correlated with salivary melatonin levels and serum CRP. Periodontal therapy resulted in a recovery of the decreased salivary melatonin levels and a negative correlation was detected for the changes of salivary melatonin and the inflammatory parameter bleeding on probing. Serum melatonin levels showed no significant differences., Conclusions: Salivary melatonin levels recovered after periodontal therapy and correlated with a decrease of local periodontal inflammation. This may imply the local involvement of melatonin in the pathogenesis of periodontitis due to its antioxidant abilities. However, the exact role of melatonin in periodontal disease remains to be investigated in future trials., Clinical Relevance: The present results suggest salivary melatonin as a risk indicator for the severity of periodontal disease.

Even though rotenone has been used extensively in recent years to produce a model of Parkinson disease in rats, its systemic effects either on neurons apart from dopaminergic structures or non-neuronal tissues have not been elucidated well. In our present study, 30 adult Sprague-Dawley rats were divided into three even groups. A short-term rotenone-treated group received 10 mg/kg b.w. rotenone daily for 7 days. The long-term rotenone-treated group received 3 mg/kg b.w. rotenone daily for 30 days. The control group received vehicle only and were kept 5 rats each in parallel to both short- and long-term rotenone treated groups. It was found that short-term rotenone treatment produced marked vascular damages associated with ischemic neuronal degeneration particularly in the thalamus, cerebellum and nucleus dentatus. In long-term rotenone-treated group, vascular changes were less severe and neuronal degeneration was associated with mild microglial proliferation and astrocytosis. Non-neuronal pathology as the result of short-term rotenone exposure consisted of degeneration and necrosis of seminiferous tubular epithelia with formation of spermatide multinucleate giant cells. On the other hand, long-term rotenone treatment did not affect testicles and only caused sinusoidal dilatation in the liver, myocardial degeneration in the heart and interstitial hemorrhages in the kidneys and lungs. In conclusions, damage to blood vasculature by rotenone appeared mediating neuronal and non-neuronal pathology in Sprague-Dawley rats. This effect might provide new insights for ethiopathogenesis of neurodegenerative diseases and contributes to the understanding of hemorrhagic stroke., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Swainsonine (SW) is an indolizidine triol plant alkaloid isolated from the species Astragalus, colloquially termed locoweed. When chronically ingested by livestock and wildlife, symptoms include severe neuronal disturbance. Toxicity to the central and peripheral nervous system is caused by inhibition of lysosomal α-mannosidase (AMA) and accumulation of intracellular oligosaccharide. Consequently, SW has been used as a model substance in investigations of lysosomal storage diseases. Involvement of the basal ganglia has been postulated due to the neuronal symptoms of affected animals. Therefore, primary midbrain cultures from embryonic mice containing dopaminergic neurons were utilized in this study. Neural cells were exposed to SW (0.01-100 μM) for 72 h. AMA activity was 50 % inhibited at 1 μM SW. Cytotoxic changes in cultures were observed above 25 μM SW by increases in lactate dehydrogenase activity and nitric oxide content. Neurotoxicity to dopaminergic cells was visualized by tyrosine hydroxylase immunohistochemistry. Structural degeneration scored as dendritic shortening and shrinkage of cell bodies was dose-dependent and resulted in nerve loss above 25 μM. SW exposure caused progression from reversible to irreversible cytotoxicity. Partial regeneration of AMA-activity in culture was observed on removal of SW. The antioxidative vitamins ascorbic acid and tocopherol (both 100 μM) partially reversed the toxic effect on dopaminergic cells and ascorbic acid decreased AMA inhibition. Thus, neuronal midbrain cell cultures can demonstrate the neurotoxic action of SW and cytoprotective strategies may be tested at a single nerve cell level.

Over recent decades, engineered nanoparticles are increasingly produced as the result of the rapid development in nanotechnology. They are currently used in a wide range of industrial and public sectors including healthcare, agriculture, transport, energy, materials, and information and communication technologies. As the result, an increasing concern has been raised over the potential impacts of engineered nanoparticles to human health. In the light of this, it is the purpose of the present review to discuss: (1) novel properties of engineered nanoparticles particularly in biomedical sciences, (2) most recently reported adverse effects of manufactured nanoparticles on human health and (3) different aspects of toxicological risk assessment of these nanoparticles., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Salivary stress-related biomarkers in connection with periodontal disease have not been extensively studied. In addition to cortisol as a well-known marker of stress loading, chromogranin A (CgA) and α-amylase (AA) are supposed to link the activity of the neuroendocrine system to local and systemic immune functions and to be related to periodontitis. This study aims to determine CgA and AA in saliva and serum in periodontal health and disease to assess their potential relationship to periodontitis., Methods: Patients with aggressive (AgP) (n = 24) and chronic periodontitis (CP) (n = 34) as well as healthy control (CO) (n = 30) individuals participated in this study. CgA and AA were determined in saliva and serum with enzyme-linked immunosorbent assay and an adapted clinical amylase test; salivary cortisol was determined using mass spectrometry. Clinical parameters of periodontal disease were evaluated, and their possible correlations with stress-related biomarkers were assessed., Results: Significantly higher CgA levels were found in the saliva of patients with AgP compared with those in patients with CP and CO individuals (P <0.001). Salivary cortisol levels were higher in the AgP group compared with those in patients with CP (P <0.05). No differences in serum CgA levels and salivary and serum AA activities were found among all groups. A positive correlation was revealed between salivary AA activity or salivary CgA levels and the extent of periodontitis (P <0.05)., Conclusion: The results suggest an association of CgA and cortisol levels as well as AA activity in saliva with periodontitis, especially a significant relationship of salivary CgA and cortisol to AgP.

Cannabinoids derived from Cannabis sativa demonstrate neuroprotective properties in various cellular and animal models. Mitochondrial impairment and consecutive oxidative stress appear to be major molecular mechanisms of neurodegeneration. Therefore we studied some major cannabinoids, i.e. delta-9-tetrahydrocannabinolic acid (THCA), delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in mice mesencephalic cultures for their protective capacities against 1-methyl-4-phenyl pyridinium (MPP(+)) toxicity. MPP(+) is an established model compound in the research of parkinsonism that acts as a complex I inhibitor of the mitochondrial respiratory chain, resulting in excessive radical formation and cell degeneration. MPP(+) (10 μM) was administered for 48 h at the 9th DIV with or without concomitant cannabinoid treatment at concentrations ranging from 0.01 to 10 μM. All cannabinoids exhibited in vitro antioxidative action ranging from 669 ± 11.1 (THC), 16 ± 3.2 (THCA) to 356 ± 29.5 (CBD) μg Trolox (a vitamin E derivative)/mg substance in the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay. Cannabinoids were without effect on the morphology of dopaminergic cells stained by tyrosine hydroxylase (TH) immunoreaction. THC caused a dose-dependent increase of cell count up to 17.3% at 10 μM, whereas CBD only had an effect at highest concentrations (decrease of cell count by 10.1-20% at concentrations of 0.01-10 μM). It influenced the viability of the TH immunoreactive neurons significantly, whereas THCA exerts no influence on dopaminergic cell count. Exposure of cultures to 10 μM of MPP(+) for 48 h significantly decreased the number of TH immunoreactive neurons by 44.7%, and shrunken cell bodies and reduced neurite lengths could be observed. Concomitant treatment of cultures with cannabinoids rescued dopaminergic cells. Compared to MPP(+) treated cultures, THC counteracted toxic effects in a dose-dependent manner. THCA and CBD treatment at a concentration of 10 μM lead to significantly increased cell counts to 123% and 117%, respectively. Even though no significant preservation or recovery of neurite outgrowth to control values could be observed, our data show that cannabinoids THC and THCA protect dopaminergic neurons against MPP(+) induced cell death., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Aim: Currently, there are almost 100 genes related to Alzheimer's disease (AD), and studies have indicated that apolipoprotein E (APO E) ε4 allele is a genetic risk factor of AD. However, there have been no reports of the distributions of APO E genotypes and allele frequencies in Uighur and Han AD patients., Methods: We analyzed APO E gene polymorphism in 209 AD cases diagnosed based on National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer's Disease and Related Disorders Association and 220 non-dementia controls. We used polymerase chain reaction-restriction fragment length polymorphism methods as the basis of this epidemiological survey., Results: In the AD and control groups, there are no statistically significant differences in APO E genotypes and allele frequency between the Uighur and Han ethnicities (P < 0.05). In the AD group, the ε3/4 genotype (28.2%) and ε4 allele frequency (14.8%) occurred at a higher rate than in the control (13.2% and 8.0%, respectively; P < 0.05). This distinction remained true within each ethnicity; the ε3/4 genotype and ε4 allele frequency are higher in the AD groups (Uighur, 30.6% and 15.8%, respectively; Han, 25.5% and 13.8%, respectively) than in the control groups (Uighur, 14.5% and 9.4%, respectively; Han, 11.7% and 6.3%, respectively; P < 0.05)., Conclusions: The distribution of APO E genotype and allele frequency does not differ between the Uighur and Han ethnicities. The APO E ε4 allele is a risk factor of AD for both populations., (© 2012 The Authors. Psychogeriatrics © 2012 Japanese Psychogeriatric Society.)

Ginsenosides are a special group of triterpenoid saponins attributed to medical effects of ginseng. Therefore, they have been research targets over the last three decades to explain ginseng actions and a wealth of literature has been presented reporting on ginsenosides' effects on the human body. Recently, there is increasing evidence on beneficial effects of ginsenosides to the central nervous system (CNS). Using a wide range of in vitro and in vivo models, researchers have attributed these effects to specific pharmacological actions of ginsenosides on cerebral metabolism, oxidative stress and radical formation, neurotransmitter imbalance and membrane stabilizing effects, and even antiapoptotic effects. Modulating these particular mechanisms by ginsenosides has thus been reported to exert either general stimulatory effects on the brain functions or protecting the CNS against various disease conditions. In this review, we try to address the recently reported ginsenosides' actions on different CNS targets particularly those supporting possible therapeutic efficacies in CNS disorders and neurodegenerative diseases., (© 2010 Blackwell Publishing Ltd.)

Background: In Parkinson's disease, most of current therapies only provide symptomatic treatment and so far there is no drug which directly affects the disease process., Objectives: To investigate the neuroprotective effects of minocycline against long-term rotenone toxicity in primary dopaminergic cell cultures., Methods: Embryonic mice of 14-days-old were used for preparation of primary dopaminergic cell cultures. On the 6th day in vitro, prepared cultures were treated both with minocycline alone (1, 5, 10 and 20 microM) and concomitantly with rotenone (5 and 20 nM) and minocycline. Cultures were incubated at 37 degrees C for six consecutive days. On Day in vitro culture medium was aspirated and used for measuring lactate dehydrogenase. Cultured cells were fixed in 4% paraformaldhyde and stained immunohistochemically against tyrosine hydroxylase., Results: Treatment of cultures with 5 and 20 nM of rotenone significantly decreased the survival of tyrosine hydroxylase immunoreactive neurons by 27 and 31% and increased the release of lactate dehydrogenase into the culture medium by 31 and 236%, respectively compared to untreated controls. Minocycline (1, 5, 10 microM) significantly protected tyrosine hydroxylase immunoreactive neurons by 17, 15 and 19% and 13, 22 and 23% against 5 and 20 nM of rotenone, respectively compared to rotenone-treated cultures. Minocycline (only at 10 microM) significantly decreased the release of lactate dehydrogenase by 79% and 133% against 5 and 20 nM of rotenone, respectively., Conclusion: Minocycline has neuroprotective potential against the progressive loss of tyrosine hydroxylase immunoreactive neurons induced by long-term rotenone toxicity in primary dopaminergic cultures.

Green tea polyphenol epigallocatechin-3-gallate (EGCG) is reported to have antioxidant abilities and to counteract beneficially mitochondrial impairment and oxidative stress. The present study was designed to investigate neuroprotective effects of EGCG on rotenone-treated dissociated mesencephalic cultures and organotypic striatal cultures. Rotenone is a potent inhibitor of complex I of the respiratory chain, which in vitro causes pathological and neurochemical characteristics of diseases in which mitochondrial impairment is involved, e.g., Parkinson's disease. Treatment with EGCG (0.1, 1, 10 muM) alone had no significant effects on mesencephalic cultures. In striatal slice cultures, EGCG led to a significant increase of propidium iodide (PI) uptake and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), but not dihydroethidium (DHE) fluorescence intensity. Rotenone (20 nM on the eighth DIV for 48 h) significantly decreased the numbers and the neurite lengths of TH ir neurons by 23 and 34% in dissociated mesencephalic cell cultures compared to untreated controls. Exposure of striatal slices to rotenone (0.5 mM for 48 h) significantly increased PI uptake, and DAF-FM and DHE fluorescence intensities by 41 and 136 and 19%, respectively, compared to controls. Against rotenone, in dissociated mesencephalic cultures, EGCG produced no significant effect on either the number or neurite lengths of THir neurons compared to rotenone-treated cultures, but EGCG significantly decreased PI uptake by 19% and DAF-FM fluorescence intensity by 19 and 58%, respectively, compared to increase in rotenone-exposed striatal slices. On the other hand, EGCG did not affect superoxide (O(2) (-)) formation as detected with DHE. These data indicate that EGCG slightly protects striatal slices by counteracting nitric oxide (NO(.)) production by rotenone. In conclusion, EGCG partially protects striatal slices but not dissociated cells against rotenone toxicity.

Thymoquinone is the main active constituent of Nigella sativa seeds with antioxidant and antiinflammatory properties. In the present study, primary dopaminergic cultures from mouse mesencephala were used to investigate the neuroprotective effects of thymoquinone against MPP(+) and rotenone toxicities. MPP(+) (10 microm on day 10 in vitro (i.v.) for 48 h) significantly decreased the number of THir by 40% compared with untreated control cultures. Rotenone at both short (20 nm on day 10 i.v. for 48 h) and long-term (1 nm on day 6 i.v. for 6 consecutive days) toxicities reduced the number of THir neurons by 33% and 24%, respectively. Treatment of cultures with thymoquinone (0.01, 0.1, 1, 10 microm on day 8 i.v. for 4 days) rescued about 25% of THir neurons at concentrations of 0.1 microm and 1 microm against MPP(+)-induced cell death. Against rotenone, thymoquinone afforded significant protection in both short- and long-term models. In short-term rotenone toxicity, thymoquinone (from days 8-12 i.v.) saved about 65%, 74% and 79% of THir neurons at concentrations of 0.01, 0.1 and 1 microm, respectively, compared with cell loss induced by rotenone. In long-term rotenone toxicity, concomitant treatment of cultures with thymoquinone significantly rescued about 83-100% of THir neurons compared with rotenone-treated cultures. In conclusion, the current study presents for the first time the potential of thymoquinone to protect primary dopaminergic neurons against MPP(+) and rotenone relevant to Parkinson's disease.

Epidemiological studies suggest the involvement of pesticides in the etiology of Parkinson's disease. Exposure to rotenone results in degeneration of the nigrostriatal pathway through inhibition of complex I. Organotypic striatal slice cultures were prepared from brains of adult mice and treated with rotenone (0.01, 0.05, 0.1, and 1 mM) for 48 h. Lactate dehydrogenase activity was elevated by 167% at 1 mM of rotenone. Using fluorescent indicators, membrane damage was up to 130% as measured by propidium iodide fluorescence, and superoxide (DHE) and nitric oxide (DAF-FM) formation were increased by 195% and 774% at 1 mM of rotenone, respectively, compared to controls. The study concludes that formation of radicals mediated striatal degeneration by rotenone.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the subsequent decrease of dopamine levels in the striatum. Epidemiological studies indicate environmental pollutants as a causative factor of sporadic PD. Experimental cell culture models have the inherent problem to mimic long-lasting neurodegeneration and to tackle its time-concentration relationship. The present study was designed to investigate the sensitivity of primary dopaminergic neurons to long-term rotenone exposure relevant to PD. Primary cultures prepared from embryonic mouse mesencephala were treated with nanomolar concentrations of rotenone (1, 3, 5, 10nM) on the 6th day in vitro (DIV) for 2, 4 and 6 days. The number of tyrosine hydroxylase immunoreactive (TH(+)) neurons and total hematoxylin-stained nuclei were counted. Astrocyte density was qualitatively evaluated by anti-glial fibrillary acidic protein (anti-GFAP) immunocytochemistry. It was found that dopaminergic neurons were highly sensitive to long-term rotenone treatment. Rotenone in a concentration- and time-dependent manner decreased the number of TH(+) neurons and led to degenerative changes of their morphology. Counting of the total cell number revealed a significant deleterious effect on the overall culture after 6 days of rotenone exposure. However, our study demonstrates a higher sensitivity of dopaminergic neurons to long-term exposure to nanomolar concentrations of rotenone. Other cells in the culture including non-dopaminergic neurons and glia cells appeared less affected compared to dopaminergic neurons.

Microdialysis technique was used to study the effects of the locally applied alpha adrenoceptor agonist phenylephrine and antagonist phentolamine on the basal noradrenaline efflux as well as on the noradrenaline uptake inhibitor desipramine-elicited noradrenaline efflux in the nucleus accumbens (NAc) of freely moving rats. Tetrodotoxin reduced basal noradrenaline efflux by 72%, whereas desipramine increased it by 204%. Phenylephrine reduced the basal noradrenaline efflux by 32% and phentolamine blocked this effect. Phentolamine elevated the basal noradrenaline efflux by 150% and phenylephrine counteracted this effect. The desipramine-elicited noradrenaline efflux was not affected by phenylephrine, but enhanced by phentolamine. Desipramine counteracted the effects of phenylephrine and potentiated those of phentolamine. These results indicate that the accumbal noradrenaline efflux is under inhibitory control of alpha adrenoceptors that are suggested to be presynaptically located on adrenergic nerve terminals in the NAc. Furthermore, this study suggests that the conformational state of alpha adrenoceptors varies across the available amount of noradrenaline. The clinical impact of these data is discussed.

A 6 years retrospective study of pesticide poisonings in domestic animals and livestock from 1999 to 2004 submitted to the Institute for Medical Chemistry, University of Veterinary Medicine, Vienna in Austria was compiled and analysed. Totally 380 pesticide analysis requests were referred by veterinary practitioners, from the Institute for Pathology of the above university, by regional and central governments as well as local police departments and district administrations, animal protectionist groups, public health authorities and private clients. Among the total number of suspected samples for pesticides, 175 (46.1%) cases were found positive to contain pesticides of various kinds. Among the pesticides found, carbamate insecticides were most prominent, representing 50.3% of the total positive cases. These compounds were followed by rodenticides-anticoagulants with 18.9% of the positive results, by organophosphate insecticides 5.1%, and by the rodenticides-nonanticoagulant 3.4%, the other 22.3% included molluscicides, herbicides, etc. In totally 225 animals, 123 animals were found positive for pesticide intoxication, among them 47.2% were dogs, 34.1% were cats 9.8% of other species and 8.9% of unspecified animal samples. The pesticides were characterized by HPLC-techniques using commercially available standards. The aim of this Austrian survey was to determine the incidence and frequency of confirmed pesticide intoxications in animals in Austria and to emphasize its relevance in veterinary practice for livestock and domestic animals.

Cytidine-5'-diphosphocholine (citicoline or CDP-choline) is an essential endogenous intermediate in the biosynthesis of phosphatidylcholine. In the present study, primary dopaminergic cultures from mouse mesencephala were treated with citicoline to investigate its neuroprotective potential on the survival of dopaminergic neurons exposed to MPP(+) and glutamate. Treatment with citicoline alone significantly increased the survival of dopaminergic neurons compared to controls. MPP(+) or glutamate decreased the total number of dopaminergic neurons whereas citicoline afforded significant protection against either toxicity. Moreover, citicoline significantly decreased propidium iodide uptake by cultured cells. The study concludes that citicoline exerts stimulant and neuroprotective actions on cultured dopaminergic neurons.

Amyloid beta (Abeta) toxicity has been implicated in cell death in the hippocampus, but its specific mechanisms are poorly understood. In this study, Abeta-induced cell death was investigated in organotypic hippocampal slice cultures (OHCs) that were cultured for various periods in vitro. There were no obvious histological differences among slices cultured for 3 to 7 weeks in vitro. Although there was little neurotoxicity after treatment with Abeta25-35 in OHCs cultured for relatively shorter periods (3-5 weeks), age-dependent cell death was evident in OHCs cultured for relatively longer periods (6-7 weeks) after exposure to Abeta25-35. In OHCs cultured for 7 weeks, S-allyl-L-cysteine (SAC), a component of aged garlic extract, protected the cells in areas CA1 and CA3 and the dentate gyrus from Abeta25-35-induced toxicity. The immunoreactivity of cleaved caspase-12 was increased whereas that of glucose-regulated protein 78 was not altered after exposure to Abeta25-35. The increases in the cleaved caspase-12 were also reversed by simultaneously applied SAC. These results suggest that OHCs cultured for relatively longer periods are more susceptible to Abeta-induced toxicity and that the Abeta-induced cell death involves caspase-12-dependent pathways. It is also suggested that SAC is able to protect against the Abeta-induced neuronal cell death through the inhibition of the caspase-12-dependent pathway.

In Parkinson's disease clinical and experimental evidence suggest that neuroinflammatory changes in cytokines caused by microglial activation contribute to neuronal death. Experimentally, neuroinflammation of dopaminergic neurons can be evoked by lipopolysaccharide (LPS) exposure. In mesencephalic primary cultures LPS (100 microg/ml) resulted in 30-50% loss of dendritic processes, changes in the perikarya, cellular atrophy and neuronal cell loss of TH-immunoreactive (TH+) cells. iNOS activity was increased dose dependently as well as prostaglandin E2 concentrations. Ginsenosides, as the active compounds responsible for ginseng action, are reported to have antioxidant and anti-inflammatory effects. Here ginsenoside Rd was used to counteract LPS neurodegeneration. Partial reduction of LPS neurotoxic action was seen in dopaminergic neurons. Cell death by LPS as well as neuroprotective action by ginsenoside Rd was not selective for dopaminergic neurons. Neuronal losses as well as cytoprotective effects were similar when counting NeuN identified neurons. The anti-inflammatory effect of ginsenoside Rd could equally be demonstrated by a reduction of NO-formation and PGE2 synthesis. Thus, protective mechanisms of ginsenoside Rd may involve interference with iNOS and COX-2 expression.

There is an ongoing controversy about potential toxicity of L-3,4-dihydroxyphenylalanine (L-dopa) to dopaminergic neurons in Parkinson's disease (PD). Neuroimaging data suggest that L-dopa accelerates the loss of dopamine nerve terminals, especially at higher doses. The disputed aspect of toxicity and the frequently observed motor complications accompanying L-dopa therapy have led to an increased use of dopamine agonists during the past two decades. Reports describing their neuroprotective potential to dopaminergic neurons have attracted much attention. Here, we describe the novel finding that the combination of a dopamine (DA) agonist, alpha-dihydroergocryptine (DHEC), with L-dopa or DA exerts a synergistic stimulatory effect on dopaminergic neurons in primary culture, while each substance alone had no or less effect. DA receptor stimulation plays a decisive role. The synergistic effect suggests that a combinatory therapy can be beneficial to slow the degeneration of dopaminergic neurons.

Oxidative stress evoked by excitotoxicity is considered an important factor for the loss of dopaminergic neurons in Parkinson's disease. In vitro, protective effects of the dopamine agonist lisuride on complex I inhibition in primary dopaminergic cell culture have been reported. However, little is known about the effects of lisuride on glutamate-induced radical formation. Here, effects of lisuride on the formation of nitric oxide (NO) and superoxide radicals following glutamate exposure were studied on primary cell cultures prepared from mouse mesencephala. Glutamate treatment resulted in doubling of NO and superoxide radical formation, increased dopaminergic cell degeneration and extensively altered neuronal appearance. Pretreatment with lisuride significantly lowered the levels of either reactive species and increased the survival of dopaminergic neurons compared to glutamate-treated cultures. Moreover, the beneficial effect of lisuride could be completely inhibited by the D2/D3 receptor antagonist sulpiride when co-treated in cultures.

Although the definite etiology of Parkinson's disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinson's disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinson's disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.

Ginseng, the root of Panax species, is a well-known herbal medicine. It has been used as a traditional medicine in China, Korea, and Japan for thousands of years and is now a popular and worldwide used natural medicine. The active ingredients of ginseng are ginsenosides which are also called ginseng saponins. Recently, there is increasing evidence in the literature on the pharmacological and physiological actions of ginseng. However, ginseng has been used primarily as a tonic to invigorate weak bodies and help the restoration of homeostasis. Current in vivo and in vitro studies have shown its beneficial effects in a wide range of pathological conditions such as cardiovascular diseases, cancer, immune deficiency, and hepatotoxicity. Moreover, recent research has suggested that some of ginseng's active ingredients also exert beneficial effects on aging, central nervous system (CNS) disorders, and neurodegenerative diseases. In general, antioxidant, anti-inflammatory, anti-apoptotic, and immune-stimulatory activities are mostly underlying the possible ginseng-mediated protective mechanisms. Next to animal studies, data from neural cell cultures contribute to the understanding of these mechanisms that involve decreasing nitric oxide (NO), scavenging of free radicals, and counteracting excitotoxicity. In this review, we focus on recently reported medicinal effects of ginseng and summarize the current knowledge of its effects on CNS disorders and neurodegenerative diseases.

Ginseng, the root of the Panax species, is a well-known herbal medicine. Traditionally it has been used in Korea, China and Japan for thousands of years. Nowadays it has become a popular and worldwide known health drug. Current scientific studies demonstrate in vivo and in vitro its beneficial effects in a wide range of pathological conditions such as cardiovascular disease, cancer, immune deficiency and hepatotoxicity. Ginsenosides or ginseng saponins as the active ingredients have antioxidant, anti-inflammatory, anti-apoptotic and immunostimulant properties, which raised speculations that these compounds could positively affect neurodegenerative disorders and delay neuronal aging. Conclusive clinical data in humans are still missing. However, results from animal studies and neuronal cell culture experiments indicate that ginsenosides can counteract and attenuate factors promoting neuronal death as environmental toxins, excitotoxic action of glutamate and rises in intracellular calcium, excessive release of free radicals and apoptotic events. Thus, neuroprotective actions of ginsenosides could come about as a valuable option to slow down neurodegenerative diseases.

Parkinson's disease (PD) is a chronic and progressive neurological disorder characterized by selective degeneration of dopaminergic neurons (DAergic) in the substantia nigra pars compacta (SNpc) and subsequent decrease in dopamine (DA) levels in the striatum. Although levodopa replacement therapy is initially effective in symptomatic treatment of parkinsonian patients, its effectiveness often declines and various levodopa-related side effects appear after long-term treatment. The disabling side effects of levodopa therapy include motor fluctuations such as the wearing-off or on-off phenomena, dyskinesias and psychiatric symptoms. Nowadays, DA receptor agonists are often regarded as first choice in de novo and young parkinsonian patients to delay the onset of levodopa therapy. In advanced stages of the disease, they are also used as adjunct therapy together with levodopa to retard the development of motor complications. DA receptor agonists mimic the endogenous neurotransmitter, dopamine, and act by direct stimulation of presynaptic (autoreceptors) and postsynaptic DA receptors. Next to their clinical role in treating parkinsonian patients, laboratory studies reported antioxidative and neuron-rescuing effects of DA receptor agonists either in vivo or in vitro. This may involve reduced DA turnover following autoreceptor stimulation and direct free radical scavenging activity. In this review, we focus on and summarize the recently reported effects of the most commonly used DA agonists either in clinical or in research studies relevant to PD treatment.

Early diagnosis of Parkinson's disease (PD) in nonsymptomatic patients is a key issue. An increased echogenicity of the substantia nigra (SN) was found previously in Parkinsonian patients and in a low percentage of healthy adults. These nonsymptomatic subjects also showed a reduced 18F-dopa uptake in striatum, suggesting a preclinical injury of the nigrostriatal system that could later proceed into PD. To investigate the ability of ultrasonography to detect markers of SN degeneration, such as iron deposition and neuromelanin depletion, we scanned postmortem brains from normal subjects at different ages by ultrasound and measured the echogenic area of the SN. The SN was then dissected and used for histological examinations and determination of iron, ferritin, and neuromelanin content. A significant positive correlation was found between the echogenic area of the SN and the concentration of iron, H- and L-ferritins. Multivariate analysis carried out considering the iron content showed a significant negative correlation between echogenicity and neuromelanin content of the SN. In PD, a typical loss of neuromelanin and increase of iron is observed in this brain area. The finding of a positive correlation between iron and ferritin levels and a negative correlation of neuromelanin content with the area of echogenicity at the SN could therefore provide an interesting basis for diagnosis and therapeutic follow-up studies in PD., (Copyright (c) 2005 Movement Disorder Society.)