carbocyanines and dihydroethidium

The present study was performed to investigate the early effects of blue light irradiation of photoreceptors in retinal explant cultures.. Murine retinal explant cultures were irradiated with visible blue light (405 nm) with an output power of 1 mW/cm2. Dihydroethidium was used to determine the production of reactive oxygen species. Morphological alterations of photoreceptor outer segments were determined by live imaging microscopy with mitochondrial dye JC-1. Transmission and scanning electron microscopy were used for ultrastructural evaluations. Cell death in the retina was assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay method.. Live retinal explants displayed an increase in reactive oxygen species production, as revealed by fluorescent dihydroethidium products in photoreceptor cells after 30 min of blue light exposure. After 3 h of exposure, blue light caused disorganization of the normally neatly stacked outer segments of living photoreceptors. Ultrastructural analysis revealed breaks in the cell membrane surrounding the outer segments, especially in the middle section. The outer segments appeared tortuous, and the lamellar structures had been disrupted. TUNEL-staining revealed that long-term blue light exposure induced photoreceptor cell death.. In vitro blue light irradiation of retinal explants is a suitable model system for investigating early ultrastructural changes, as well as damage that leads to cell death in photoreceptor cells.

Topics: Animals; Benzimidazoles; Carbocyanines; Cell Death; Ethidium; Female; Fluorescent Dyes; In Situ Nick-End Labeling; Light; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Organ Culture Techniques; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Retina

Topics: Animals; Carbocyanines; Deuterium; Ethidium; Fluorescent Dyes; Kinetics; Mice; Optical Imaging; Oxidation-Reduction; Superoxides

The neurotoxin 6-hydroxydopamine is commonly used in models of Parkinson's disease, and a potential factor in the pathogenesis of the disease. However, the mechanisms responsible for 6-hydroxydopamine-induced dopaminergic degeneration have not been totally clarified. Reactive oxygen species (ROS) derived from 6-OHDA uptake and intraneuronal autooxidation, extracellular 6-OHDA autooxidation, and microglial activation have been involved. The mitochondrial implication is controversial. Mitochondrial ATP-sensitive K (mitoK(ATP)) channels may provide a convergent target that could integrate these different mechanisms. We observed that in primary mesencephalic cultures and neuron-enriched cultures, treatment with the mitoK(ATP) channel blocker 5-hydroxydecanoate, inhibits the dopaminergic degeneration induced by low doses of 6-OHDA. Furthermore, 5-hydroxydecanoate blocks the 6-OHDA-induced decrease in mitochondrial inner membrane potential and inhibits 6-OHDA-induced generation of superoxide-derived ROS in dopaminergic neurons. The results suggest that low doses of 6-OHDA may generate low levels of ROS through several mechanisms, which may be insufficient to induce neuron death. However, they could act as a trigger to activate mitoK(ATP) channels, thereby enhancing ROS production and the subsequent dopaminergic degeneration. Furthermore, the present study provides additional data for considering mitoK(ATP) channels as a potential target for neuroprotection.

Topics: Adrenergic Agents; Analysis of Variance; Animals; Antihypertensive Agents; Benzimidazoles; Carbocyanines; Cell Count; Cells, Cultured; Decanoic Acids; Diazoxide; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Ethidium; Glyburide; Hydroxy Acids; Membrane Potential, Mitochondrial; Mesencephalon; Neurons; Oxidopamine; Potassium Channel Blockers; Potassium Channels; Rats; Reactive Oxygen Species; Rhodamines; Time Factors; Tritium; Tyrosine 3-Monooxygenase

Leishmania spp. are intracellular protozoa residing in mononuclear phagocytes. Leishmania organisms are susceptible to microbicidal responses generated in response to phagocytosis. Assuming that both phagocyte and parasite populations are heterogeneous, it is advantageous to examine the response of individual cells phagocytosing living parasites. Because Leishmania spp. lose virulence during the raising of transfectants, we developed a method to label live Leishmania chagasi short-term with fluorescent dyes. Up to six parasite divisions were detected by flow cytometry after labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE), dioctadecyl-tetramethylindo carbocyanine perchlorate, or chloromethyl tetramethylrhodamine. Labeled parasites entered mononuclear phagocytes as determined by confocal and time-lapse microscopy. Dihydroethidium (DHE) was used to detect macrophage-derived oxidants generated during phagocytosis. Presumably Leishmania organisms are opsonized with host serum/tissue components such as complement prior to phagocytosis. Therefore, we investigated the effects of opsonization and found that this increased the efficiency of CFSE-labeled parasite entry into monocytes (84.6% +/- 8.8% versus 20.2% +/- 3.8% monocytes infected; P < 0.001). Opsonization also increased the percentage of phagocytes undergoing a respiratory burst (66.0% +/- 6.3% versus 41.0% +/- 8.3% of monocytes containing CFSE-labeled parasites; P < 0.001) and the magnitude of oxidant generation by each infected monocyte. Inhibitor data indicated that DHE was oxidized by products of the NADPH oxidase. These data suggest that opsonized serum components such as complement lead to more efficient entry of Leishmania into their target cells but at the same time activate the phagocyte oxidase to generate microbicidal products in infected cells. The parasite must balance these positive and negative survival effects in order to initiate a viable infection.

Topics: Animals; Carbocyanines; Cells, Cultured; Ethidium; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Humans; Leishmania infantum; Microscopy, Confocal; Microscopy, Video; Monocytes; Opsonin Proteins; Oxidants; Phagocytes; Rhodamines; Staining and Labeling; Succinimides

Polyunsaturated fatty acids (PUFAs) can influence tumor growth and migration, both in vitro and in vivo. The PUFA gamma-linolenic acid (GLA) has been reported to improve the poor prognosis associated with human gliomas, although its effects at sublethal concentrations on residual cells postsurgery are poorly understood. The study investigated the effects sublethal PUFA doses (90 or 150 microM) may have on rat C6 glioma cell energy metabolism, since an adequate energy supply is essential for cell proliferation, migration, and apoptosis. Of note was the identification of mitochondrial heterogeneity in relation to the mitochondrial membrane potential (MMP), which has been suggested but unproven in previous studies. GLA and eicosapentaenoic acid (EPA) caused significant changes in cellular fatty acid composition and increased the percentage of cells with a low MMP after a 96-h exposure period. The presence of PUFAs inhibited C6 cell proliferation and migration, although apoptosis was not induced. The protein expression and activity of glucose-6-phosphate dehydrogenase was increased after 96-h incubation with 90 microM GLA and EPA and would allow redox regulation through increased NADPH production, permitting the maintenance of adequate intracellular reduced glutathione concentrations and limiting rates of lipid peroxidation and reactive oxygen species generation. Neither NADP(+)-isocitrate dehydrogenase nor NADP(+)-malate dehydrogenase activity responded to PUFAs, suggesting it is glucose-6-phosphate dehydrogenase that is the principal source of NADPH in C6 cells. These data compliment studies showing that higher concentrations of GLA induced glioma cell death and tumor regression and suggest that GLA treatment could be useful for the inhibition of residual cell proliferation and migration after surgical removal of the tumor mass.

Topics: Animals; Annexin A5; Benzimidazoles; Blotting, Western; Carbocyanines; Cell Movement; Cells, Cultured; Cytochromes c; DNA Fragmentation; Ethidium; Fatty Acids, Unsaturated; Fluorescent Antibody Technique; Fluorescent Dyes; Gas Chromatography-Mass Spectrometry; Glioma; Glucosephosphate Dehydrogenase; Membrane Potentials; Microscopy, Confocal; Mitochondria; Mitosis; Rats; Time Factors