Ultrastructure of Hepatocytes from Laboratory Mice Fed a Standard Dry Laboratory Animal Diet.

The significant destructive changes in ultrastructure of hepatocytes from laboratory mice kept in different vivariums in Moscow and fed with dry laboratory animal diets acquired from different domestic manufacturers that were not standardized for initial products were demonstrated using electron microscopy. Furthermore, disruption in the ultrastructure of liver parenchymal cells occurred regardless of the animal status (SPF or conventional), conditions of various vivariums, as well as the feed manufacturer. At the same time, studies on ultrastructure of liver hepatocytes from mice kept in the Charles River Laboratory facilities in Germany and fed with the Altromin Spezialfutter laboratory animal diet (GmbH & Co., Germany) that was produced using quality control of ingredients did not reveal destructive changes in the internal ultrastructure of hepatocytes. However, if these mice were later fed with the food produced in local manufactures, changes in the structure of liver cells developed after 2 months. Thus, feeding with dry diet from the domestic producers of an unspecified composition causes significant changes in the ultrastructure of hepatocytes in control animals, reflecting the development of some pathological processes in the body.

Nonphosphorylating Oxidation in Mitochondria and Related Processes.

The mechanism of oxidative phosphorylation and its regulation remain one of the main problems of bioenergetics. Efficiency of the mitochondrial energization is determined by the relationship between the rate of generation of electrochemical potential of hydrogen ions and the rate of its expenditure on the synthesis of ATP and the use of ATP in endergonic reactions. Uncoupling (partial or complete), which occurs in the process of uncontrolled and controlled leakage of ions through the inner mitochondrial membrane, on the one hand leads to the decrease in the relative synthesis of ATP, and on the other, being consistent with the law of conservation of energy, leads to the formation of heat, generation of which is an essential function of the organism. In addition to increased thermogenesis, the increase of non-phosphorylating oxidation of various substrates is accompanied by the decrease in transmembrane potential, production of reactive oxygen species, and activation of oxygen consumption, water and carbon dioxide production, increase in the level of intracellular ADP and acidification of the cytosol. In this analysis, each of these factors will be considered separately for its role in regulating metabolism.

Mitochondria in Obliquely Striated Muscles of the Horsehair Worm Gordionus alpestris (Nematomorpha, Gordioidea) with Structural Organization Typical of Cells with Energy-Intensive Processes.

The ultrastructure of mitochondria in the flattened circomyarian fibers of the horsehair worm Gordionus alpestris (Nemathelminthes) was examined. In contrast to the previously published data, we showed these mitochondria to be giant elongated organelles that densely fill the central cytoplasmic space of the ribbon-like muscle fibers. No fundamental differences were found in the ultrastructure of the muscle tissue mitochondria in actively moving free-living and parasitic G. alpestris worms. The functional significance of the observed ultrastructural organization of mitochondria is discussed in connection with the necessity for an extended mitochondrial membrane system for a uniform supply of active muscle tissue with energy.

Study of Age-Dependent Structural and Functional Changes of Mitochondria in Skeletal Muscles and Heart of Naked Mole Rats (Heterocephalus glaber).

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per µm2 of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles - from 0.21 ± 0.01 to 0.29 ± 0.03 µm2. Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.

Age-Related Changes in Ultrastructure of Mitochondria. Effect of SkQ1.

For many years, investigators have attempted to identify unique ultrastructural conditions of mitochondria related to aging. However, this did not result in definitive results. At present, this issue has again become of topical interest due to development of the mitochondrial theory of aging and of engineering of a novel antioxidant class known as mitochondria-targeted antioxidants. The review briefly discusses experimental results that, from our perspective, allow the most objective understanding regarding age-related changes in mitochondrial ultrastructure.

Morphometric Examination of Mitochondrial Ultrastructure in Aging Cardiomyocytes.

Mitochondrial ultrastructure in cardiomyocytes from 3- and 24-month-old Wistar and OXYS rats was investigated using a new approach designed for morphometric analysis. The data fully confirm the electron microscopy data: the area of the inner mitochondrial membrane per unit volume of mitochondria was significantly decreased with age, as found on heart muscle section. In 3-month-old Wistar rats from the control group, this parameter was 41.3 ± 1.52 µm(2)/µm(3), whereas in OXYS rats it was decreased to 30.57 ± 1.74 µm(2)/µm(3). With age, an area of the inner mitochondrial membrane per unit volume of mitochondria declined in both rat strains: Wistar - from 41.3 ± 1.52 to 21.47 ± 1.22 µm(2)/µm(3), OXYS - from 30.57 ± 1.74 to 16.3 ± 0.89 µm(2)/µm(3). A new method that we designed and used for morphometric analysis notably simplifies the process of morphometric measurements and opens up good opportunities for its further optimization using image recognition technology.

Ultrastructural changes in ageing lacrimal gland in Wistar rats.

We studied age-related ultrastructural reorganization in acinar cells and intercalary ducts of the lacrimal gland acini in 3-, 15-, and 24-month-old Wistar rats. Ultrastructural changes in the lacrimal gland progressed with age and led to dramatic ultrastructural reconstruction of the lacrimal gland at the age of 24 months. These changes mainly included complete destruction of acinar cells and increase in the number of enlarged branched ducts that filled the greater part of gland volume; these dusts were lined with epithelial cells with altered ultrastructure. Acinar cells in the acini communicate via special connecting intermembrane complexes formed by desmosomes and mitochondria adjacent to them in each contacting cell. It is assumed that association of mitochondria with desmosomes found in the acini is a special functional complex indicating that every single acinus is a functional formation. This assumption is indirectly confirmed by the fact that the destruction never occurred in a single cell, but always involved all cells constituting the acini. The revealed ultrastructural changes reflect age-related deterioration of the secretory function of the lacrimal gland.

[Structural and functional basis for accelerated thymic involution in OXYS rats].

Thymus involution is one of the most pronounced manifestations of aging immune system, associated with increase susceptibility to infections, autoimmune diseases and cancer. Its nature in normal aging is actively under investigation. Much less attention is paid to the study of mechanisms of accelerated thymic involution. Previously, we showed the connection of accelerated senescence in OXYS rats with accelerated thymic involution, the mechanisms of which remain unclear. The aim of the present work was to study multifunctional condition of thymic epithelial cells in aging OXYS rats. Immunohistochemical analysis showed the reduction of thymic epithelial cell net in OXYS rats, significant decrease in volume and surface area of epithelial cells in cortical substance as compared to control Wistar rats. Electron microscopic study revealed the marked changes of epithelial cell ultrastructure, namely the reduction of cytoplasm volume, sharp decrease in size and quantity of secretory vacuoles, the presence of multiple autophagosomes and phagolysomes. The results indicate that one of the possible mechanisms of epithelial cell net reduction in thymus of senescence-accelerated OXYS rats can be the aggravation of autophagy, probably associated with mitochondrial dysfunction typical for OXYS rats. Despite the known fact of slowing autophagy with aging in some tissues, the example of OXYS rats allows to suggest that chronic deviation of intensity of this process from physiological level, either to decrease or activation, can lead to degenerative changes in organs and finally form the progeric phenotype of the whole organism.

SkQ1 slows development of age-dependent destructive processes in retina and vascular layer of eyes of wistar and OXYS rats.

We show the development of clearly pronounced age-related pathological changes in eye tissues of Wistar and OXYS rats. Photoreceptor cells were virtually absent in all OXYS rats in the age of 24 months. Massive accumulations of lipofuscin granules were detected in the pigmented epithelium cells. Flattening, overgrowing, and degradation of endothelial cells of choriocapillaries were also observed. Along with these changes, vessels without signs of degradation were detected in the pigmented epithelium. In 24-month-old Wistar rats these changes were local and were seen in only some of the animals. The mitochondria-targeted antioxidant SkQ1 (the rats were given SkQ1 daily with food at the dose of 250 nmol/kg for 5 months, starting from the age of 19 months) prevented the development of these pathological changes in both Wistar and OXYS rats. The data were subjected to mathematical processing and statistical analysis.

Lipofuscin granule dynamics during development of age-related macular degeneration.

The pigment epithelium cell structure and therapeutic effect of antioxidant SkQ1, selectively penetrating into mitochondria from eye drops, were studied upon development in OXYS rats of age-related retinopathy as a model of macular degeneration. The characteristic dynamics and ultrastructural peculiarities of the layer of electron-dense cytoplasmic structures of the pigment epithelium apex part and incorporated lipofuscin granules were revealed. The therapy of OXYS animals for 68 days using 250 nM SkQ1 drops decreased the extent of development of age-related macular degeneration. Electron-microscopic investigation showed that SkQ1 prevented development of ultrastructural changes in the pigment epithelium characteristic of macular degeneration, the condition of which after therapy with SkQ1 drops corresponded to ultrastructure of pigment epithelium in Wistar rats of the same age having no symptoms of retinal damage. It is supposed that ultrastructural changes in the electron-dense layer upon development of age-related macular degeneration are indicative of disturbances in the optical cycle functioning, especially of disturbances in functioning of photoreceptor membranes.

[Yeast cell ultrastructure after amiodarone treatment].

[Amiodarone is used as a pharmaceutical substance for treating a number of diseases. However it is known that structural and functional disturbances are caused by amiodarone in patient's tissues. Here particular features of amiodarone effect are studied in yeast Saccharomyces cerevisiae, where amiodarone was shown to cause apoptosis. Electron-microscopic study of yeast cells after amiodarone treatment reveals a significant increase in lipid particle number which can lead to formation of a structural complex by interacting with membranous organelles of a cell. Amiodarone causes the appearance of small and separated slightly swollen mitochondria. Chro-matin displacement to the periphery of nucleus, nuclear sectioning and nuclear envelope disturbances are observed in the cells under these conditions. The detected cell ultrastructure alterations in the S. cerevisiae are considered to be specific response to the phospholipidosis and apoptosis caused by amiodarone.

[The cytochrome c oxidase activity in mitochondria of cardiomyocytes of isolated cardiac tissue under long-term hypoxic incubation].

Using method of electron microscopic histochemistry based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to reveal cytochrome c oxidase activity we identified that long hypoxic incubation of isolated small pieces of cardiac tissue during 72 h caused changes in mitochondrial ultrastructure followed by a breach of functional activities of mitochondria, and, in particular, complex IV of the respiratory chain. But for all that, small electron-dense mitochondria appearing inside electron-light mitochondria ("mitochondria inside mitochondria") stained positively for cytochrome c oxidase activity along the full length of cristaes. The results obtained are discussed in connection with conception of changes in the mitochondrial reticulum ultrastructure during mitoptosis.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: synthesis and in vitro studies.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke).

Effects of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6'-plastoquinonyl) decylrhodamine 19 (SkQR1) on rat models of H2O2- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H2O2 or by ischemia/reperfusion. Higher SkQ1 (125-250 nmol/kg per day for 2-3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2-4 days, whereas one injection of SkQ1 or SkQR1 (1 micromol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 micromol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals.

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age-induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 microM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 5. SkQ1 prolongs lifespan and prevents development of traits of senescence.

Very low (nano- and subnanomolar) concentrations of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) were found to prolong lifespan of a fungus (Podospora anserina), a crustacean (Ceriodaphnia affinis), an insect (Drosophila melanogaster), and a mammal (mouse). In the latter case, median lifespan is doubled if animals live in a non-sterile vivarium. The lifespan increase is accompanied by rectangularization of the survival curves (an increase in survival is much larger at early than at late ages) and disappearance of typical traits of senescence or retardation of their development. Data summarized here and in the preceding papers of this series suggest that mitochondria-targeted antioxidant SkQ1 is competent in slowing down execution of an aging program responsible for development of age-related senescence.

Mitochondrial matrix fragmentation as a protection mechanism of yeast Saccharomyces cerevisiae.

It was shown that separate fragments of the inner mitochondrial compartment (mitoplasts) can exist under a single non-fragmented outer membrane. Here we asked whether fragmentation of the inner mitochondria could prevent rupturing of the outer membrane and release of pro-apoptotic molecules from the mitochondrial intermembrane space into the cytoplasm during mitochondrial swelling. First, we showed that in Saccharomyces cerevisiae yeast addition of amiodarone causes formation of electrically separate compartments within mitochondrial filaments. Moreover, amiodarone treatment of Deltaysp2 mutant produced a higher proportion of cells with electrically discontinuous mitochondria than in the wild type, which correlated with the survival of cells. We confirmed the existence of separated mitoplasts under a single outer membrane using electron microscopy. Mitochondria with fragmented matrixes were also detected in cells of the stationary phase. Our data suggest that such fragmentation acts as a cellular protective mechanism against stress.

[Dynamics of mitochondrial ultrastructure in small pieces of cardiac tissue under long-term anoxic incubation].

Anoxic incubation of isolated small pieces of cardiac tissue for 72 h caused emergence of an unusual population of mitochondria, referred to as "mitochondrion inside mitochondrion". We studied dynamics of the origin of this event. In the most part of a mitochondrial population after a 6 h anoxic incubation of myocardial tissue, a local increase in some region of the intermembrane space was observed. Some regions of matrix with adjoined inner membrane move into these regions of intermembrane space, to be constricted eventually. After 12 h of incubation densely neighbouring layers of membrane are observed in these structures. By 24 h of incubation, inside new-formed structures well-distinguished concentric layers of membrane appear. Between these layers some electron-dense material ultrastructurally identical to mitochondrial matrix is seen. By 72 h of anoxic incubation, in cardiomyocytes of the experimental tissue structures with well-marked morphological features of mitochondria appear, which we called "mitochondrion inside mitochondrion". Results of our study are discussed in terms of a conception of changes that occur in the structure of mitochondrial reticulum during apoptosis.

Programmed cell death in plants: effect of protein synthesis inhibitors and structural changes in pea guard cells.

Pea leaf epidermis incubated with cyanide displayed ultrastructural changes in guard cells that are typical of apoptosis. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, and lincomycin, an inhibitor of protein synthesis in chloroplasts and mitochondria, produced different effects on the dynamics of programmed death of guard cells. According to light microscopy data, cycloheximide reinforced and lincomycin suppressed the CN(-)-induced destruction of cell nuclei. Lincomycin lowered the effect of cycloheximide in the light and prevented it in the dark. According to electron microscopy data, the most pronounced effects of cycloheximide in the presence of cyanide were autophagy and a lack of apoptotic condensation of nuclear chromatin, the prevention of chloroplast envelope rupturing and its invagination inside the stroma, and the appearance of particular compartments with granular inclusions in mitochondria. Lincomycin inhibited the CN(-)-induced ultrastructural changes in guard cell nuclei. The data show that programmed death of guard cells may have a combined scenario involving both apoptosis and autophagy and may depend on the action of both cytoplasm synthesized and chloroplast and mitochondrion synthesized proteins.

Effect of "external" superoxide anion on apoptosis in coleoptiles of wheat seedlings.

A derivative of phthalic acid, dibutylphthalate (DBP), which has gametocidal effect at the concentration of approximately 10(-4) M, increased apoptosis in coleoptiles of wheat seedlings. This was associated with activation of chromatin margination and generation of mitochondria-containing vesicles. At the same concentration, DBP activated the release by the coleoptiles of superoxide anion into the environment. Lower (10(-5) M) and higher (10(-3) M) concentrations of DBP virtually had no effect on either process. A probable mechanism of effect of the "external" superoxide anion on apoptosis within the plant cell is discussed.