Intensity Of DNA Fragmentation Research Articles

Comparative influence of mesenchymal stromal cells of different origin on DNA fragmentation of neuronal nuclei during ischemia-reperfusion of the somatosensory cortex of the rat brain

Relevance: One of the main causes of stroke in acute cerebrovascular accident (ACVA) is ischemia, which begins with the formation of an acute neuronal energy deficit with subsequent activation of the "ischemic cascade" reactions that lead to irreversible damage to nervous tissue. Aim: To compare the effect of mesenchymal stromal cells (MSCs) of different origin and human MSCs from Wharton's jelly lysate on neuroapoptotic changes in the somatosensory cortex of the rat brain in conditions of model ischemia-reperfusion (IR) performed by ductal cytoflowmetry. Materials and methods: The experiment was carried out using 165 four-month-old male Wistar rats weighing 160-190 g, which were subjected to bilateral 20-minute transient ischemia-reperfusion (IR) of the internal carotid arteries. After modeling the pathology, the animals were injected into the femoral vein (iv) with MSCs obtained from umbilical cord Wharton jelly, human and rat adipose tissue in the amount of 106 cells/animal. Other groups of experimental animals were intravenously injected with fetal rat fibroblasts in the amount of 106 cells/animal (in 0.2 ml of physiological solution) and MSCs from umbilical cord Wharton's jelly lysate in a dose of 0.2 ml/animal. Control animals were injected intravenously with 0.2 ml of physiological solution. The level of DNA fragmentation in the nuclei of neurons of the somatosensory cortex of rats on the 7th day after ischemia-reperfusion was studied by flow cytometry. The research was carried out on a flow cytometer "Partech РАС" of the company Partech, Germany. The statistical significance of the differences was assessed by Student's t-test. Results: The study noted an increase in the level of fragmented DNA in a group of animals with IR by 3.25 times 7 days after model IR. The performed treatment showed that in groups with transplanted MSCs of various origins and MSC lysate from human Wharton's jelly cells, the intensity of DNA fragmentation in the nuclei of neurons in rat brain somatosensory cortex reliably decreased in1.8-2. 6 times compared with the group of control pathology (IR without treatment). Conclusions: Experimental 20-minute IR of the brain of rats forms a persistent focus of necrotic and apoptotic death of neurons, which is manifested by an increase in fragmented DNA (3.25 times). Intravenous transplantation of MSCs of various origin and lysate of MSCs from human Wharton jelly has a therapeutic effect in model IR, which is manifested by a decrease in the processes of neuro-destruction and neuroapoptosis in the area of ischemic brain damage Such effect is a link to the polytrophic mechanism of MSCs neuro-protective action.

Metformin mitigates amyloid β1-40-induced cognitive decline via attenuation of oxidative/nitrosative stress and neuroinflammation.

Metformin is an antidiabetic medicine widely used for management of type 2 diabetes with neuroprotective effects and promising potential to attenuate cognitive impairment. The efficacy of metformin in attenuation of Alzheimer's disease (AD) pathology has not been well-documented. Thus, this study was designed to assess protective effect of metformin against Aβ1-40-instigared cognitive impairment. After intra-CA1 microinjection of aggregated Aβ1-40, rats received oral metformin (50 and/or 200mg/kg/day) for two weeks. Cognition function was analyzed in various behavioral tasks besides measurement of hippocampal oxidative stress, apoptosis, and inflammation along with H&E staining and 3-nitrotyrosine (3-NT) immunohistochemistry. Obtained data showed significant improvement of discrimination score in novel object recognition test, higher alternation score in Y maze, greater latency in passive avoidance task, and lower working and reference memory errors in radial arm maze in metformin-treated Aβ-injured group. Moreover, metformin treatment attenuated hippocampal levels of nitrite, MDA, protein carbonyl, ROS, TNFα, GFAP, DNA fragmentation intensity, caspase 3 activity, AChE activity, and increased SOD activity and level of IL-10 as an anti-inflammatory factor. In addition, metformin treatment was associated with lower CA1 neuronal loss and it also decreased intensity of 3-NT immunoreactivity as an indicator of nitrosative stress. Taken together, obtained findings showed neuroprotective and anti-dementia property of metformin in male rats and this may have potential benefit in attenuation of cognitive decline and related complications in patients with neurodegenerative disorders such as AD besides diabetes mellitus.

The Cytotoxicity, DNA Fragmentation, and Decreasing Velocity Induced By Chromium(III) Oxide on Rainbow Trout Spermatozoa.

The present study aimed to determine the cytotoxicity of chromium(III) oxide micro particles (Cr2O3-Ps) in rainbow trout (Oncorhynchus mykiss) spermatozoa. Firstly, Cr2O3-Ps were synthesized and structurally characterized the surface, morphological for particle size and thermal properties. In addition, its structural and elemental purity was determined using energy-dispersive X-ray (EDX) spectrum and elemental maps. Structural purity, thermal properties, and stability of Cr2O3-Ps were also examined in detail by performing thermal analysis techniques. The cytotoxicity of Cr2O3-Ps was measured by the observation of velocities, antioxidant activities, and DNA damages in rainbow trout spermatozoa after exposure during 3h in vitro incubation. The straight line velocity (VSL), the curvilinear velocity (VCL), and the angular path velocity (VAP) of spermatozoa decreased after exposure to Cr2O3-Ps. While the superoxide dismutase (SOD) and the catalase (CAT) decreased, the lipid peroxidation increased in a dose-dependent manner. However, the total glutathione (tGSH) was not affected in this period. DNA damages were also determined in spermatozoa using Comet assay. According to DNA in tail (%) data, DNA damages have been detected with gradually increasing concentrations of Cr2O3-Ps. Furthermore, all of class types which are categorized as the intensity of DNA fragmentation has been observed between 50 and 500µg/L concentrations of Cr2O3-Ps exposed to rainbow trout spermatozoa. At the end of this study, we determined that the effective concentrations (EC50) were 76.67µg/L for VSL and 87.77µg/L for VCL. Finally, these results about Cr2O3-Ps may say to be major risk concentrations over 70µg/L for fish reproduction in aquatic environments.

Chronic alcoholism-mediated metabolic disorders in albino rat testes.

There is good evidence for impairment of spermatogenesis and reductions in sperm counts and testosterone levels in chronic alcoholics. The mechanisms for these effects have not yet been studied in detail. The consequences of chronic alcohol consumption on the structure and/or metabolism of testis cell macromolecules require to be intensively investigated. The present work reports the effects of chronic alcoholism on contents of free amino acids, levels of cytochrome P450 3A2 (CYP3A2) mRNA expression and DNA fragmentation, as well as on contents of different cholesterol fractions and protein thiol groups in rat testes. Wistar albino male rats were divided into two groups: I – control (intact animals), II – chronic alcoholism (15% ethanol self-administration during 150 days). Following 150 days of alcohol consumption, testicular free amino acid content was found to be significantly changed as compared with control. The most profound changes were registered for contents of lysine (–53%) and methionine (+133%). The intensity of DNA fragmentation in alcohol-treated rat testes was considerably increased, on the contrary CYP3A2 mRNA expression in testis cells was inhibited, testicular contents of total and etherified cholesterol increased by 25% and 45% respectively, and protein SH-groups decreased by 13%. Multidirectional changes of the activities of testicular dehydrogenases were detected. We thus obtained complex assessment of chronic alcoholism effects in male gonads, affecting especially amino acid, protein, ATP and NADPH metabolism. Our results demonstrated profound changes in testes on the level of proteome and genome. We suggest that the revealed metabolic disorders can have negative implication on cellular regulation of spermatogenesis under long-term ethanol exposure.

Acute Antiapoptotic Effects of Hydrocortisone in the Hippocampus of Neonatal Rats

Natural glucocorticoid hydrocortisone was suggested as a potent substitution for dexamethasone in the treatment of bronchopulmonary dysplasia in neonates. The aim of this study was to investigate whether hydrocortisone is able to affect the expression of apoptotic genes and the intensity of naturally occurring cell death in the developing rat hippocampus. Hormone treatment decreased procaspase-3 and active caspase-3 levels as well as DNA fragmentation intensity in the hippocampal formation of one-week-old rats in 6 h after injection. These changes were accompanied by an upregulation of antiapoptotic protein Bcl-XL, while expression of proapoptotic protein Bax remained unchanged. The action of hydrocortisone was glucocorticoid receptor-independent, as the selective glucocorticoid receptor agonist dexamethasone did not affect either apoptotic protein levels or DNA fragmentation intensity in the hippocampal region. The data are the first evidences for in vivo antiapoptotic effects of hydrocortisone in the developing hippocampus.

Involvement of apoptosis during deciduomal regression in pseudopregnant hamsters Effect of progesterone

We determined whether fragmentation of genomic DNA, apoptosis, occurs during deciduomal regression in pseudopregnant hamsters and the effect of progesterone on the apoptotic processes. Artificially induced deciduoma were obtained on different days of pseudopregnancy and separated into mesometrial and antimesometrial tissues. The deciduomal cell cycle progression and population profiles of both sides were compared by flow cytometry. The proportion of sub-G 1 peak, which was correlated with the apoptotic cells, were about 10% on day 8 and reached to 40% in both tissues on day 10. Exogenous progesterone treatment by daily injection (2 mg; s.c.) on and after day 8 reduced the percentage of low molecular weight DNA in both tissues on day 10 and day 12 as compared to the non-treated control one, respectively. The appearance of DNA ladder was also delayed at least 24 h by progesterone administration. The intensity of DNA fragmentation was more pronounced in antimesometrial deciduoma. In situ 3′-end labeling of apoptotic cells further substantiated the apoptotic process. The apoptotic cells first appeared in the luminal region in antimesometrial deciduoma on day 8 and spreaded all over the entire deciduomal tissue on day 10. Progesterone treatment stimulated deciduomal proliferating cell nuclear antigen (PCNA) expression, maintained deciduoma until day 14 and retarded the differentiation and regeneration of the uterine epithelium.

Comments on "Radiation-Induced Apoptosis in HL60 Cells: Oxygen Effect, Relationship between Apoptosis and Loss of Clonogenicity, and Dependence of Time to Apoptosis on Radiation Dose" by Hopcia et al. (Radiat. Res. 145, 315-323, 1996)

irradiation with doses as high as 20 Gy induced almost no apoptosis within 6 h although irradiation with 5 Gy induced apoptosis in almost half of the cells by 3-4 days. Nevertheless, the authors concluded that the oxygen enhancement ratios (OERs) for apoptosis in HL60 cells at 6 h and 3 days after irradiation were similar to that for the radiationinduced decline in clonogenicity of the cells. The authors deserve a compliment for their effort to determine the OER for apoptosis, which has never been done before. However, we would like to point out that some of the results described in the article differ substantially from the observations reported by other investigators. In the study by Hopcia et al., HL60 cells were very radioresistant so that, in the estimation of the OER for apoptosis at 6 h after irradiation, the oxygenated and hypoxic HL60 cells had to be irradiated with doses as high as 80 Gy and 160 Gy, respectively. We wonder why the HL60 cells used by Hopcia et al. were so resistant to radiation-induced apoptosis while most normal as well as malignant lymphoid cells, including HL60 cells, have been reported to undergo apoptosis within several hours after an exposure to relatively mild external stress including X irradiation, UV irradiation and hyperthermia (2-5). In our previous study we observed that HL60 cells undergo apoptosis in 3 h after heating at 42'C for 60 min (6). The results shown in Figs. 1 and 2 in the paper by Hopcia et al. indicate that the level of apoptosis determined 6 h after irradiation with doses as high as 20 Gy was hardly greater than the apoptosis in the unirradiated control cells. Such a resistance to radiation-induced apoptosis in HL60 cells is, as Hopcia et al. themselves pointed out, distinctly different from the report by Abend et al. (2) that marked apoptosis occurred in HL60 cells within 24 h after an exposure to 2.0-2.5 Gy X rays. As described below, we also observed that significant apoptosis occurs in HL60 cells within several hours after irradiation with doses higher than 2 Gy. Our results on the agarose gel electrophoresis of DNA extracted from HL60 cells 4 h after irradiation are shown in Fig. 1. It can be seen that irradiation with 4 Gy X rays caused a faint but visible ladder composed of DNA fragments, indicating that the cells underwent apoptosis. The intensity of DNA fragmentation increased as the dose was inc eased up to 12 Gy. We repeated the same experiments more than five times and always obtained similar results. Results of our flow cytometry study are shown in Fig. 2. In this particular study, the percentage of apoptotic HL60 cells before irradiation was 4.5% and it increased to 47.3% 4 h