Identification of Metabolomic Signatures for Ischemic Hypoxic Encephalopathy Using a Neonatal Rat Model.

A study was performed to determine early metabolomic markers of ischemic hypoxic encephalopathy (HIE) using a Rice-Vannucci model for newborn rats. Dried blood spots from 7-day-old male and female rat pups, including 10 HIE-affected animals and 16 control animals, were analyzed by liquid chromatography coupled with mass spectrometry (HPLC-MS) in positive and negative ion recording modes. Multivariate statistical analysis revealed two distinct clusters of metabolites in both HPLC-MS modes. Subsequent univariate statistical analysis identified 120 positive and 54 negative molecular ions that exhibited statistically significant change in concentration, with more than a 1.5-fold difference after HIE. In the HIE group, the concentrations of steroid hormones, saturated mono- and triglycerides, and phosphatidylcholines (PCs) were significantly decreased in positive mode. On the contrary, the concentration of unsaturated PCs was increased in the HIE group. Among negatively charged molecular ions, the greatest variations were found in the categories of phosphatidylcholines, phosphatidylinositols, and triglycerides. The major metabolic pathways associated with changed metabolites were analyzed for both modes. Metabolic pathways such as steroid biosynthesis and metabolism fatty acids were most affected. These results underscored the central role of glycerophospholipid metabolism in triggering systemic responses in HIE. Therefore, lipid biomarkers' evaluation by targeted HPLC-MS research could be a promising approach for the early diagnosis of HIE.

Unique Features of the Tissue Structure in the Naked Mole Rat (Heterocephalus glaber): Hypertrophy of the Endoplasmic Reticulum and Spatial Mitochondrial Rearrangements in Hepatocytes.

The reason for the exceptional longevity of the naked mole rat (Heterocephalus glaber) remains a mystery to researchers. We assumed that evolutionarily, H. glaber acquired the ability to quickly stabilize the functioning of mitochondria and endoplasmic reticulum (ER) to adjust metabolism to external challenges. To test this, a comparison of the hepatic mitochondria and ER of H. glaber and C57BL/6 mice was done. Electron microscopy showed that 2-months-old mice have more developed rough ER (RER) than smooth ER (SER), occupying ~17 and 2.5% of the hepatocytic area correspondingly, and these values do not change with age. On the other hand, in 1-week-old H. glaber, RER occupies only 13% constantly decreasing with age, while SER occupies 35% in a 1-week-old animal, constantly rising with age. The different localization of mitochondria in H. glaber and mouse hepatocytes was confirmed by confocal and electron microscopy: while in H. glaber, mitochondria were mainly clustered around the nucleus and on the periphery of the cell, in mouse hepatocytes they were evenly distributed throughout the cell. We suggest that the noted structural and spatial features of ER and mitochondria in H. glaber reflect adaptive rearrangements aimed at greater tolerance of the cellular system to challenges, primarily hypoxia and endogenous and exogenous toxins. Different mechanisms of adaptive changes including an activated hepatic detoxification system as a hormetic response, are discussed considering the specific metabolic features of the naked mole rat.

LC-MS Analysis Revealed the Significantly Different Metabolic Profiles in Spent Culture Media of Human Embryos with Distinct Morphology, Karyotype and Implantation Outcomes.

In this study we evaluated possible differences in metabolomic profiles of spent embryo culture media (SECM) of human embryos with distinct morphology, karyotype, and implantation outcomes. A total of 153 samples from embryos of patients undergoing in vitro fertilization (IVF) programs were collected and analyzed by HPLC-MS. Metabolomic profiling and statistical analysis revealed clear clustering of day five SECM from embryos with different morphological classes and karyotype. Profiling of day five SECM from embryos with different implantation outcomes showed 241 significantly changed molecular ions in SECM of successfully implanted embryos. Separate analysis of paired SECM samples on days three and five revealed 46 and 29 molecular signatures respectively, significantly differing in culture media of embryos with a successful outcome. Pathway enrichment analysis suggests certain amino acids, vitamins, and lipid metabolic pathways to be crucial for embryo implantation. Differences between embryos with distinct implantation potential are detectable on the third and fifth day of cultivation that may allow the application of culture medium analysis in different transfer protocols for both fresh and cryopreserved embryos. A combination of traditional morphological criteria with metabolic profiling of SECM may increase implantation rates in assisted reproductive technology programs as well as improve our knowledge of the human embryo metabolism in the early stages of development.

Progressive reorganization of mitochondrial apparatus in aging skeletal muscle of naked mole rats (Heterocephalus glaber) as revealed by electron microscopy: potential role in continual maintenance of muscle activity.

The authors examined the ultrastructure of mitochondrial apparatus of skeletal muscles of naked mole rats (Heterocephalus glaber) from the age of 6 months to 11 years. The obtained results have demonstrated that the mitochondria in skeletal muscles of naked mole rats aged below 5 years is not well-developed and represented by few separate small mitochondria. Mitochondrial reticulum is absent. Starting from the age of 5 years, a powerful mitochondrial structure is developed. By the age of 11 years, it become obvious that the mitochondrial apparatus formed differs from that in the skeletal muscle of adult rats and mice, but resembles that of cardiomyocytes of rats or naked mole rats cardiomyocytes. From the age of 6 months to 11 years, percentage area of mitochondria in the skeletal muscle of naked mole rat is increasing by five times. The growth of mitochondria is mainly driven by increased number of organelles. Such significant growth of mitochondria is not associated with any abnormal changes in mitochondrial ultrastructure. We suppose that specific structure of mitochondrial apparatus developed in the skeletal muscle of naked mole rats by the age of 11 years is necessary for continual skeletal muscle activity of these small mammals burrowing very long holes in stony earth, resembling continual activity of heart muscle. In any case, ontogenesis of naked mole rat skeletal muscles is much slower than of rats and mice (one more example of neoteny).

N-Acyl Dopamines Induce Apoptosis in Endometrial Stromal Cells from Patients with Endometriosis.

Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N-acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N-arachidonoyldopamine, N-docosahexaenoyldopamine, and N-oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.

Mitochondria in the Nuclei of Rat Myocardial Cells.

Electron microscopic study of cardiomyocytes taken from healthy Wistar and OXYS rats and naked mole rats (Heterocephalus glaber) revealed mitochondria in nuclei that lacked part of the nuclear envelope. The direct interaction of mitochondria with nucleoplasm is shown. The statistical analysis of the occurrence of mitochondria in cardiomyocyte nuclei showed that the percentage of nuclei with mitochondria was roughly around 1%, and did not show age and species dependency. Confocal microscopy of normal rat cardiac myocytes revealed a branched mitochondrial network in the vicinity of nuclei with an organization different than that of interfibrillar mitochondria. This mitochondrial network was energetically functional because it carried the membrane potential that responded by oscillatory mode after photodynamic challenge. We suggest that the presence of functional mitochondria in the nucleus is not only a consequence of certain pathologies but rather represents a normal biological phenomenon involved in mitochondrial/nuclear interactions.

Delayed Onset of Age-Dependent Changes in Ultrastructure of Myocardial Mitochondria as One of the Neotenic Features in Naked Mole Rats (Heterocephalus glaber).

In this study, the ultrastructure of mitochondria in cardiomyocytes of naked mole rats (Heterocephalus glaber) aged from 6 months to 11 years was examined. Mitochondria in cardiomyocytes of naked mole rats have a specific ultrastructure that is different from those in cardiomyocytes of other mammalian species studied to date. In contrast to mitochondria of other mammalian cardiomyocytes, where the internal space is completely filled by tightly packed parallel rows of cristae, mitochondria in cardiomyocytes of naked mole rats have a chaotic pattern of cristae organization with wave-like contours. Gradual formation of mitochondrial ultrastructure occurs in naked mole rats for many years. Two mitochondrial populations are developed to the age of 5 years. In addition to the main population, there are some large organelles which exceed normal sizes by two to three times. Most cristae in these mitochondria are assembled into small groups, which form the curved and ring-like structures. The appearance of some specific structural changes (i.e. bundles of parallel cristae) is observed in the mitochondrial population of naked mole rat after 11 years of age. However, these bundles are very rare and of sporadic nature. Morphometric analysis has shown that the superficial density of the inner mitochondrial membrane is similar in all examined age groups of naked mole rats: 21.1 at 6 months; 23.21 at 3 years; 23.55 at 5 years; and 20.8 at 11 years. This level is almost two times lower than in other animals studied (mice and rats). The data demonstrate that pathological changes in mitochondrial apparatus are not present in naked mole rats at least until the age of 11 years. The mitochondrial apparatus corresponds to the phenotype in young animals, thus being another neotenic feature in naked mole rats.

Mitochondria-targeted antioxidant SkQ1 reduces age-related alterations in the ultrastructure of the lacrimal gland.

Dry eye syndrome is an eye disorder affecting many people at an old age. Because dry eye syndrome is accelerated by aging, a useful approach to the prevention of this syndrome may be an intervention into the aging process. Previously, we showed that the mitochondria-targeted antioxidant SkQ1 delays manifestations of aging and inhibits the development of age-related diseases including dry eye syndrome. Nevertheless, the link between SkQ1's effects and its suppression of age-related changes in the lacrimal gland remains unclear. Here we demonstrated that dietary supplementation with SkQ1 (250 nmol/[kg body weight] daily) starting at age 1.5 months significantly alleviated the pathological changes in lacrimal glands of Wistar rats by age 24 months. By this age, lacrimal glands underwent dramatic deterioration of the ultrastructure that was indicative of irreversible disturbances in these glands' functioning. In contrast, in SkQ1-treated rats, the ultrastructure of the lacrimal gland was similar to that in much younger rats. Morphometric analysis of electron-microscopic specimens of lacrimal glands revealed the presence of numerous secretory granules in acinar cells and a significant increase in the number of operating intercalary ducts. Our results confirm that dietary supplementation with SkQ1 is a promising approach to healthy ageing and to prevention of aberrations in the lacrimal gland that underlie dry eye syndrome.

Antioxidant SkQ1 delays sarcopenia-associated damage of mitochondrial ultrastructure.

A comparative electron-microscopic study of ultrastructure of mitochondria in skeletal muscles of the 3- and 24-month-old Wistar and OXYS rats revealed age-dependent changes in both general organization of the mitochondrial reticulum and ultrastructure of mitochondria. The most pronounced ultrastructure changes were detected in the OXYS rats suffering from permanent oxidative stress. In the OXYS rats, significant changes in mitochondrial ultrastructure were detected already at the age of 3 months. Among them, there were the appearance of megamitochondria and reduction of cristae resulting in formation of cristae-free regions inside mitochondria. In the 24-month-old OXYS rats, mitochondrial reticulum was completely destroyed. In the isotropic region of muscle fiber, only small solitary mitochondria were present. There appeared regions of lysed myofibrils as well as vast regions filled with autophagosomes. A mitochondrial antioxidant SkQ1 (given to rats with food daily in the dose of 250 nmol/kg of body weight for 5 months beginning from the age of 19 months) prevented development of age-dependent destructive changes in both the Wistar and OXYS rats.