Mechanochemical Synergism of Reactive Oxygen Species Influences on RBC Membrane.

The influences of various factors on blood lead to the formation of extra reactive oxygen species (ROS), resulting in the disruption of morphology and functions of red blood cells (RBCs). This study considers the mechanisms of the mechanochemical synergism of OH• free radicals, which are most active in the initiation of lipid peroxidation (LPO) in RBC membranes, and H2O2 molecules, the largest typical diffusion path. Using kinetic models of differential equations describing CH2O2t and COH•t, we discuss two levels of mechanochemical synergism that occur simultaneously: (1) synergism that ensures the delivery of highly active free radicals OH• to RBC membranes and (2) a positive feedback system between H2O2 and OH•, resulting in the partial restoration of spent molecules. As a result of these ROS synergisms, the efficiency of LPO in RBC membranes sharply increases. In blood, the appearance of OH• free radicals is due to the interaction of H2O2 molecules with free iron ions (Fe2+) which arise as a result of heme degradation. We experimentally established the quantitative dependences of COH• CH2O2 using the methods of spectrophotometry and nonlinear curve fitting. This study extends the analysis of the influence of ROS mechanisms in RBC suspensions.

Structural Configuration of Blood Cell Membranes Determines Their Nonlinear Deformation Properties.

The ability of neutrophils and red blood cells (RBCs) to undergo significant deformations is a key to their normal functioning. Disruptions of these processes can lead to pathologies. This work studied the influence of structural configuration rearrangements of membranes after exposure to external factors on the ability of native membranes of neutrophils and RBCs to undergo deep deformation. The rearrangement of the structural configuration of neutrophil and RBC membranes under the influence of cytological fixatives caused nonlinear deformation phenomena. There were an increase in Young's modulus, a decrease in the depth of homogeneous bending, and a change in the distance between cytoskeletal junctions. Based on the results of the analysis of experimental data, a mathematical model was proposed that describes the process of deep bending of RBСs and neutrophil membranes.

Topological Relationships Cytoskeleton-Membrane Nanosurface-Morphology as a Basic Mechanism of Total Disorders of RBC Structures.

The state of red blood cells (RBCs) and their functional possibilities depend on the structural organization of the membranes. Cell morphology and membrane nanostructure are compositionally and functionally related to the cytoskeleton network. In this work, the influence of agents (hemin, endogenous oxidation during storage of packed RBCs, ultraviolet (UV) radiation, temperature, and potential of hydrogen (pH) changes) on the relationships between cytoskeleton destruction, membrane nanostructure, and RBC morphology was observed by atomic force microscope. It was shown that the influence of factors of a physical and biochemical nature causes structural rearrangements in RBCs at all levels of organization, forming a unified mechanism of disturbances in relationships "cytoskeleton-membrane nanosurface-cell morphology". Filament ruptures and, consequently, large cytoskeleton pores appeared. The pores caused membrane topological defects in the form of separate grain domains. Increasing loading doses led to an increase in the number of large cytoskeleton pores and defects and their fusion at the membrane nanosurfaces. This caused the changes in RBC morphology. Our results can be used in molecular cell biology, membrane biophysics, and in fundamental and practical medicine.

Two-step process of cytoskeletal structural damage during long-term storage of packed red blood cells.

BACKGROUND: Storage of packed red blood cells (PRBC) for 42 days causes morphological, structural, and functional changes in the red cells. To assess the quality of stored PRBC, it is important to evaluate the main components of the product. The aim of this study was to evaluate the kinetics of the structural transformations in the cytoskeleton of red cells during long-term storage (up to 42 days). MATERIALS AND METHODS: Bags of PRBC were stored with CPD/SAGM solution at +4 °C. Cytoskeletal parameters were measured on days 3, 12, 19, 21, 24, 28, 35, and 42 of storage to determine their changes. Atomic force microscopy was used to obtain images and analyse the parameters of the cytoskeletal network. As the storage time increased, a general PRBC test was performed. Membrane fixatives were not used at any stage of the preparation of the specimens for cytoskeletal imaging. RESULTS: When PRBC were stored for 42 days, the main changes to the cytoskeletal mesh included rupture of filaments, merger of small pores into larger ones, a decrease of the number of pores, thickening of filaments, and an increase of membrane stiffness. A process of irreversible changes to the cytoskeleton started on days 19-21. A kinetic model of changes in the parameters of the cytoskeletal mesh with time of PRBC storage was created. DISCUSSION: Two stages of impairment in cytoskeletal elements were found: rupture of filaments and clustering of protein components. The typical time of development and specifics of these stages are discussed. The consequences of the altered configuration of the cytoskeleton are also discussed. Destruction of the red cell cytoskeleton can have a negative effect on the efficacy of blood transfusion and increase the risk of post-transfusion complications. Our findings can be used in clinical medicine to evaluate the quality of PRBC for blood transfusion as well as for studies of the molecular organisation of red cells undergoing various types of physical and chemical treatment.

The relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs.

BACKGROUND AND OBJECTIVES: In clinical practice, it has been shown that transfusion of packed red blood cells (pRBCs) with late shelf life increases the risk of post-transfusion complications. OBJECTIVE: To study relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs. MATERIALS AND METHODS: pRBCs were processed and stored according to blood bank procedure, for 42 days, at +4°C; pRBC samples were taken on days 3, 12, 19, 21, 24, 28, 35 and 42. Cytoskeleton images and membrane stiffness were studied using atomic force microscope. RESULTS: In the course of the pRBC storage, the cytoskeleton network configuration underwent structural changes. Simultaneously, pRBC membrane stiffness was increasing, with the correlation coefficient 0·88. Until 19 days, the stiffness grew slowly, in 19-24 days there occurred a transition period, after which its growth rate was three times higher than the initial. A chain of pathological processes developed in pRBC during long storage: pH reduction (linked to increased oxidative stress), then cytoskeletal destruction and an associated increase in pRBC membrane stiffness. CONCLUSION: During prolonged storage of pRBCs and their acidification, there is a progression of pRBC cytoskeletal changes and associated increase of membrane stiffness, observed to increase in rate after days 19-24. Mutual measurements of cytoskeletal integrity and membrane stiffness may be useful quality assessment tool to study the molecular mechanisms of RBC structural degradation during storage.

Transformation of membrane nanosurface of red blood cells under hemin action.

Hemin is the product of hemoglobin oxidation. Some diseases may lead to a formation of hemin. The accumulation of hemin causes destruction of red blood cells (RBC) membranes. In this study the process of development of topological defects of RBC membranes within the size range from nanoscale to microscale levels is shown. The formation of the grain-like structures in the membrane ("grains") with typical sizes of 120-200 nm was experimentally shown. The process of formation of "grains" was dependent on the hemin concentration and incubation time. The possible mechanism of membrane nanostructure alterations is proposed. The kinetic equations of formation and transformation of small and medium topological defects were analyzed. This research can be used to study the cell intoxication and analyze the action of various agents on RBC membranes.

Opposite effects of electroporation of red blood cell membranes under the influence of zinc ions.

The goal of the study was to investigate the effects of zinc ions of various concentrations on the nanostructure of membrane of red blood cells in in vitro experiment. The suspension of red blood cells extracted from whole human blood was used. The calibrated electroporation and the atomic force microscopy (AFM) were used to analyse damage to membrane nanostructure. We studied the haemolysis after the electroporation at different zinc concentrations. A low concentration of zinc (0.15-0.5 mM) increased significantly the rate of haemolysis and reduced the residual level of non-haemolyzed cells. At high concentrations of zinc ions (0.5-10 mM), the rate constant was sharply reduced, at the same time the residual level increased. The relationship between haemoglobin coagulants and the zinc concentration was examined. High concentration of zinc caused haemoglobin aggregation. It was shown by AFM that the membrane nanostructure was essentially changed. It was experimentally established that there existed a special point of zinc concentration C = 0.5±0.1 mM at which the course of the conjugate processes on the membranes of red blood cells was changed.

Comparison of red blood cell membrane microstructure after different physicochemical influences: atomic force microscope research.

PURPOSE: After the influence of different actions on the blood, the erythrocytes may change their macrostructure. At the same time, the microstructure of cell membrane will be changed as well. This study provides the results of comparison of red blood cell membrane microstructure after they have been affected by different factors. MATERIALS AND METHODS: Images and spatial profiles of the cell surface were obtained by atomic force microscope. It was proposed to use spatial Fourier transform to decompose the initial complex profile into series of simple ones. This made it possible to compare surface parameters after exposure of red blood cells to different external actions. RESULTS: Quantitative differences between membrane profile harmonic composition parameters (amplitude and spatial period) after physical impact (impulse electrical field, osmotic swelling) and after chemical impact (the fixing fluid glutaraldehyde and the drug Esmeron) were experimentally confirmed. CONCLUSIONS: Such experimental and theoretical approach may lay down the foundations of mechanisms of different factors' effect on red blood cells both in research and in clinics.

Protection of mice against X-ray injuries by the post-irradiation administration of guanosine and inosine.

PURPOSE: To examine the radioprotective action of guanosine (Guo) and inosine (Ino) administered to mice after irradiation with X-rays. MATERIALS AND METHODS: Survival of mice exposed to lethal and sublethal doses of X-rays was studied. Peripheral blood cells were counted using a light microscope. The damage to bone marrow cells was assessed by micronucleus (MN) test. Damage and repair of DNA in blood leukocytes were estimated using the comet assay. RESULTS: Mice injected intraperitoneally (i.p.) with Guo or Ino ( approximately 30 microg g(-1), i.e., approximately 0.6 mg per 20-g mouse) 15 min after acute whole-body irradiation with 7 Gy recovered from X-ray injury. On the 30th day after irradiation, 50 and 40% of mice injected with Guo and Ino, respectively, remained alive. The dose reduction factor (DRF) was 1.23 for Guo and 1.15 for Ino. The protective effect gradually decreased as the time interval between the irradiation and injection was increased to 3, 5, 8 h. Guo and Ino facilitated the restoration of peripheral blood cell counts. These compounds protected bone marrow cells from damage and normalized erythropoiesis. Guo and Ino contributed to a more rapid and complete repair of DNA in mouse leukocytes irradiated both in vitro and in vivo. CONCLUSION: Guo and Ino introduced shortly after irradiation reduce leukopenia and thrombocytopenia and offer promise as therapeutic agents for treatment of radiation injuries.

DNA damage in frog erythrocytes after in vitro exposure to a high peak-power pulsed electromagnetic field.

Till the present time, the genotoxic effects of high peak-power pulsed electromagnetic fields (HPPP EMF) on cultured cells have not been studied. We investigated possible genotoxic effects of HPPP EMF (8.8 GHz, 180 ns pulse width, peak power 65 kW, repetition rate 50 Hz) on erythrocytes of the frog Xenopus laevis. We used the alkaline comet assay, which is a highly sensitive method to assess DNA single-strand breaks and alkali-labile lesions. Blood samples were exposed to HPPP EMF for 40 min in rectangular wave guide. The specific absorption rate (SAR) calculated from temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The temperature rise in the blood samples at steady state was 3.5 +/- 0.1 degrees C. The data show that the increase in DNA damage after exposure of erythrocytes to HPPP EMF was induced by the rise in temperature in the exposed cell suspension. This was confirmed in experiments in which cells were incubated for 40 min under the corresponding temperature conditions. The results allow us to conclude that HPPP EMF-exposure at the given modality did not cause any a-thermal genotoxic effect on frog erythrocytes in vitro.