Erythrocyte Shape Changes Research Articles

Multi-shape erythrocyte deformability analysis by imaging technique

In their extensive circulation through the cardiovascular circuit, erythrocytes are forced to transform and assume different shapes depending on the varying flow conditions in different blood vessels. The present work aims to visualize various erythrocyte shape transformations by an in vitro microcirculatory model, and to visualize multi-shape erythrocyte deformability. The model used an in-house fabricated and inexpensive disposable micro flow channel to mimic certain in vivo conditions, and a fast frame rate video microscopic system to visualize the shape changes in erythrocytes. Results have shown the multi-shape transformation of erythrocytes as resting discoidal shape, asymmetrically deformed ‘hat’ and ‘bullet-like’ shapes, and axially deformed ‘slipper- and spindle-like’ shapes. Specific erythrocytes demonstrated shape transition and transformation while passing through the observed window. The obtained erythrocyte shaper were thoroughly analyzed based on the deformability index. Findings from the image processing techniques were significant (P<0.001) for the different shapes compared with the resting shape. The simple glass in-house fabricated microfludic channel allowed for multi-shape erythrocyte deformability characterization, many of which manifest in human circulation.

Low-Level Mercury Can Enhance Procoagulant Activity of Erythrocytes: A New Contributing Factor for Mercury-Related Thrombotic Disease

BackgroundAssociations between cardiovascular diseases and mercury have been frequently described, but underlying mechanisms are poorly understood.ObjectivesWe investigate the procoagulant activation of erythrocytes, an important contributor to thrombosis, by low-level mercury to explore the roles of erythrocytes in mercury-related cardiovascular diseases.MethodsWe used freshly isolated human erythrocytes and ex vivo and in vivo thrombosis models in rats to investigate mercury-induced procoagulant activity.ResultsProlonged exposure to low-dose mercuric ion (Hg2+; 0.25–5 μM for 1–48 hr) induced erythrocyte shape changes from discocytes to echinocytes to spherocytes, accompanied by microvesicle (MV) generation. These MVs and remnant erythrocytes expressed phosphatidylserine (PS), an important mediator of procoagulant activation. Hg2+ inhibited flippase, an enzyme that recovers PS into the inner leaflet of the cell membrane, and activated scramblase, an enzyme that alters lipid asymmetry in the cell membrane. Consistent with these activity changes, Hg2+ increased intracellular calcium and depleted ATP and protein thiol. A thiol supplement reversed Hg2+-induced MV generation and PS exposure and inhibited the increase in calcium ion (Ca2+) and depletion of ATP, indicating that free-thiol depletion was critical to Hg2+-mediated procoagulant activity. The procoagulant activity of Hg2+-treated erythrocytes was demonstrated by increased thrombin generation and endothelial cell adhesion. We further confirmed Hg2+-mediated procoagulant activation of erythrocytes in ex vivo and in vivo rat thrombosis models, where Hg2+ treatment (0.5–2.5 mg/kg) increased PS exposure and thrombus formation significantly.ConclusionThis study demonstrated that mercury could provoke procoagulant activity in erythrocytes through protein-thiol depletion–mediated PS exposure and MV generation, ultimately leading to enhanced thrombosis.

Laser interference microscopy in erythrocyte study

With the laser interference microscopy (LIM) technique, one can measure phase height of cells—a variable proportional to the cell thickness and the difference in the refractive indices of the cell and the surrounding medium. This makes functional optical cell imaging possible, and estimation of shape, thickness, and area of erythrocytes feasible. In this paper, we studied changes in erythrocyte shape and volume with osmolarity and pH. Obtained from the LIM technique, erythrocyte phase heights and area values, as well as the hematocrit-measured erythrocyte volume, were used to estimate changes in the refractive index with osmolarity and pH. A comparison between the estimated refractive index with the refractive index, calculated in the assumption that it can only depend on the hemoglobin concentration in the cell, indicates that these two estimates are identical in the range of osmolarity (250–1000 mOsm) and pH (4.5–10.0) values. Thus, refractive index changes result exclusively from the changes in hemoglobin concentration with the changes in erythrocyte volume. Under these conditions, it is possible to estimate the amount of hemoglobin in an erythrocyte from its phase height and area, obtained from LIM.

Development of cell hypoxia induced by factors of long-term spaceflight

308 Changes in erythrocyte shape and plasma membrane viscosity and selective permeability were studied as well as the content of oxygen and the efficiency of its retention by human erythrocytic hemoglobin after a long-term spaceflight (LSF) were determined. It has been found that 24 h after LSF, the discocyte count decreases, the plasma membrane viscosity and the activities of Na + /H + exchange and Ca 2+ -dependent a + channel increase, Ca 2+ -ATPase activity and the hemoglobin content decrease, and the efficiency of O 2 retention by hemoglobin increases. It is assumed that the detected changes are the cellular factors that are involved in hypoxia development in humans during LSF. LSF factors (zero gravity, the action of electromagnetic fields and radiation, hypodynamia, and psychological stress) cause integrated changes in the red blood lineage of cosmonauts [1‐6]. Decrease in the erythrocyte count and hemoglobin content accompanied by an increase in the count of transformed erythrocytes, changes in the plasma membrane permeability and cell metabolism, increase in the relative cholesterol concentration, and decrease in the level of phospholipids in the plasma membrane have been recorded [2, 7]. A decrease in the ATP content both during and after LSF is associated with the activation of active ion transport, possible changes in the state of plasma membrane, and altered hemoglobin synthesis. It is important that the LSF factors cause changes in both the hemoglobin composition (increase in the fraction of A 2 hemoglobin and methemoglobin and decrease in the activity of methemoglobin reductase) and physicochemical properties of globin (shift in hemoglobin isoelectric point to the alkaline region) [6]. Characteristic changes in the erythrocyte shape caused by the LSF factors have been detected. More precisely, the specific morphological features of the erythrocyte include changes in the diameter, hemoglobin content and concentration, and cell thickness and volume. Erythrocyte has the shape of a biconcave disc, which is optimal for the oxygen diffusion into the cell. In the case of spherical erythrocytes, the central part of the cell is less saturated with oxygen than the surface layers. In addition, erythrocyte in this case loses the ability to rapidly and reversibly change its configuration to pass freely through the spleen capillaries and sinuses. This may considerably influence its lifespan [6, 7]. The decreased count of discocytes and increased counts of transformed erythrocyte forms (spherocytes, stomatocytes, knizocytes, and echinocytes) is observed during the first days after LSF [2]. It has been found that the average erythrocyte volume, thickness, and specificity increase after LSF, whereas the average surface area decreases. LSF influences not only the cell shape, but also the electrophoretic mobility of hemoglobin molecule, which is likely to be accompanied by changes in the protein redistribution in the cytoplasm. We have earlier demonstrated that characteristic changes in the erythrocyte shape and volume as well as the cell plasma membrane viscosity and permeability

Electrospun Chitosan‐Coated Fibers of Poly(L‐lactide) and Poly(L‐lactide)/Poly(ethylene glycol): Preparation and Characterization

Fibrous poly(L-lactide) (PLLA) and bicomponent PLLA/poly(ethylene glycol) mats were prepared by electrospinning and then were coated with chitosan. The presence of chitosan coating was proved by scanning electron microscopy and by fluorescence microscopy. On contact with blood, the chitosan coating led to changes in erythrocyte shape and in their aggregation. The haemostatic activity of the mats increased with increasing chitosan content. Microbiological studies against Staphylococcus aureus revealed that the chitosan coating imparts antibacterial activity to the hybrid mats. The combined haemostatic and antibacterial activities render these novel materials suitable for wound-healing applications.

Erythrocyte Shape Change Prevents Plasmodium falciparum Invasion

Normal erythrocytes have biconcave discoid shape that presents large surface area with higher cell surface to volume ratio than that of spherical shape. This appears to allow membrane internalization required for Plasmodium falciparum (Pf) invasion into erythrocytes. Indeed abnormal erythrocyte shape with decreased surface area to volume ratio such as hereditary spherocytosis limits invasion of the parasite. In the present study, using several agents to induce erythrocyte shape changes, we examined whether echinocytic shape change with membrane projections in opposite direction to membrane internalizations and/or stomatocytic shape change with decreased surface area to volume ratio that would be required for internalization, prevent Pf invasion. Having microscopically confirmed echinocytic and/or stomatocytic shape changes and also measured extensibility using an ektacytometer of the treated cells, subsequent Pf invasion assay was performed and parasitaemia determined. Both sodium flouride (NaF) and phospholipase A2 (PLA2) induced echinocytic change whereas phospholipase D (PLD), sphingomyelinase (SMase) and chlorpromazine (CPZ) caused stomatocytic change with decreased extensibility of erythrocytes. In both situations, Pf invasion was prevented, indicating that biconcave discoid shape of normal erythrocytes with high surface to volume ratio is required for membrane internalization when Pf invades into erythrocytes.

Ultrastructure of Babesia WA1 (Apicomplexa: Piroplasma) During Infection of Erythrocytes in a Hamster Model

Babesia Washington-1 (WA1) is a newly identified intraerythrocyte infectious agent of human babesiosis in the western United States. The purpose of the present study is to describe the ultrastructural changes in affected erythrocytes during the infectious process in a susceptible animal model, the golden Syrian hamster. Two, 1-mo-old female hamsters were inoculated intraperitoneally (i.p.) with 1.8 x 10(9) Babesia WA1-infected erythrocytes originally isolated from a human case and serially passaged in hamsters. Saphenous vein blood samples (20 microl) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 hr postinoculation (PI). Parasitemia was determined at each time interval by quick staining of blood smears showing 0, 2.5, 5, 10, 12.5, 22.5, 70, and almost 100% parasitemic erythrocytes at the corresponding PI time interval, respectively. Animals showed weakness and dehydration 72 hr PI inoculation, and were killed by 96 hr PI. Selected blood samples from 0, 24, 48, 72, and 96 hr were fixed in cacodylate buffer, dehydrated in ethanol gradients, resin embedded, and then thin sectioned and stained with uranyl acetate and lead citrate for transmission electron microscopy or gold-coated for scanning electron microscopy (SEM). Shape and surface membrane changes in erythrocytes were demonstrated by SEM and were more evident at 72 and 96 hr PI. Infected erythrocytes underwent changes in shape 24 hr PI, from few protrusions to several perforations, some of them resembling a "swiss cheese" appearance 96 hr PI. Several erythrocytes had irregular surface membranes and Babesia WA1 organisms were seen at different stages of development within erythrocytes, from single trophozoites to several merozoites (young trophozoites), some of them dividing to form typical tetrads. In general, Babesia WAI induced severe morphological changes in the erythrocytes, and these changes were more evident in almost all infected cells 96 hr PI.

PECULIARITIES OF POIKILOCYTOSIS INDUCED BY REACTIVE NITROGEN SPECIES ACTION

The changes in a shape, a structure and mechanical properties of erythrocyte membrane after the treatment of whole human blood with peroxynitrite were studied by the methods of light, electron scanning and atomic force microscopy. The primary mechanisms of the changes in erythrocyte shape (acanthocytosis and spherocytosis) at the action of reactive nitrogen species state to be lipid phase separation as a result of lipid peroxidation and spectrin aggregation.

Structural effects of titanium citrate on the human erythrocyte membrane

The structural effects of titanium citrate on the human erythrocyte membrane were studied through its interaction with intact erythrocytes and isolated unsealed human erythrocyte membranes (IUM). The studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Titanium citrate induced shape changes in erythrocytes, which were damaged and ruptured leaving empty and retracted membranes. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar head group and the acyl chain packing arrangements of the membrane phospholipid bilayer. Titanium citrate also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that titanium citrate induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC, while the effects on DMPE bilayers were negligible. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles. All these findings indicate that the structural perturbations induced by titanium to human erythrocytes can be extended to other cells, thereby affecting their functions.

Use of Cell Transit Analyser pulse height to study the deformation of erythrocytes in microchannels

Information on microvascular rheology can be used to develop mathematical models of network hemodynamics in various contexts: tumor-induced angiogenesis, reaction to ischemia and collateralization, hypertension, … The rheological method which probably most closely simulates red blood cell (RBC) flow behavior in the microcirculation is the determination of individual erythrocyte transit time and shape changes during flow through cylindrical micropores. However, these filtration experiments remain difficult to interpret in terms of cell flow properties, because the shape of the cell inside the pore is unknown. This paper uses the cell transit analyser (CTA) electrical pulse (especially the pulse height) to determine the relationship between the cell velocity and deformed shape in the pore, depending on the flow strength, suspending medium viscosity and cell membrane elasticity. As predicted by published numerical simulations for the flow of deformable particles through narrow channels, increasing pressure leads to increased deformation, but for a given pressure, increased viscosity leads to a slight increase in deformation. The sensitivity of filtration experiments to cell mechanical properties is improved when using low driving pressures and narrow and sufficiently long pores.

Cadmium-induced changes in the membrane of human erythrocytes and molecular models

The structural effects of cadmium on cell membranes were studied through the interaction of Cd 2+ ions with human erythrocytes and their isolated unsealed membranes (IUM). Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Cd 2+ induced shape changes in erythrocytes, which took the form of echinocytes. According to the bilayer couple hypothesis, this result meant that Cd 2+ ions located in the outer monolayer of the erythrocyte membrane. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar headgroup and the acyl chain packing arrangements of the membrane phospholipid bilayer. Cd 2+ ions also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers the erythrocyte membrane, respectively. X-ray diffraction indicated that Cd 2+ ions induced structural perturbation of the polar headgroup and of the hydrophobic acyl regions of DMPC, while the effects of cadmium on DMPE bilayers were much milder. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles (LUV). All these findings point to the important role of phospholipid bilayers in the interaction of cadmium on cell membranes.

Influence of PAMAM dendrimers on human red blood cells

Polyamidoamine (PAMAM) dendrimers with different concentrations (1 nM–1 mM) (generations 2, 3, and 4) impact on human red blood cell morphology, and membrane integrity is studied. Erythrocyte shape changes from biconcave to echinocytic in dendrimers. Cell aggregation occurs. Polymers cause also concentration- and generation-dependent haemolysis.

Human Red Blood Cells: Rheological Aspects, Uptake, and Release of Cytotoxic Drugs

The shape of a normal human red blood cell (RBC) is well known: under resting conditions it is that of a biconcave discocyte. However, RBCs can easily undergo transformation to other shapes with stomatocytes and echinocytes as extremes. Various anticancer agents, generally reactive and labile substances, e.g., oxazaphosphorines and fluoropyrimidines, can induce severe deformation of shape. Shape changes in erythrocytes can induce rheological disturbances, which occasionally have pathophysiological consequences. It is difficult to estimate the impact of shape changes on the in vivo behavior of agents of biological interest. However, it has been demonstrated for various anticancer agents that erythrocytes fulfill an important role in their uptake, transport, and release. Moreover, some anticancer agents are capable of influencing important transporters such as MRP and GLUT-1. Monitoring of erythrocyte concentrations of certain cytotoxic agents is therefore of interest as the data generated can have a predictive outcome for therapeutic efficacy. This is true for cyclophosphamide, ifosfamide, lometrexol, and 6-mercaptopurine, as well as MRP and GLUT-1 mediated agents.

Urea fragments drug treated human erythrocytes by externalization without temperature increase

Application of an appropriate amount of urea or urea with drug into a suspension of human erythrocytes in microcapillaries incubated with or without crenating or cup-forming drug in 5mM Hepes buffered saline, pH 7.4, at room temperature (25oC), resulted in the incorporation of urea into the membrane and induction of cell shape change of echinocytic type and fragmented the cells by externalization without causing haemolysis. The time to completion of urea induced cell fragmentation by externalization phenomenon was not influenced by drug concentration. It is suggested that urea was not an alternative to heat for the study of drug membrane interaction when cytoskekeleton are reduced. Based on these results, the effects of exposure of human erythrocytes to urea environment are discussed. Keywords: urea- therapeutic use; fertilizer production environment, human erythrocytes, Running title: Erythrocyte shape change induced by urea. (Global Journal of Pure and Applied Sciences: 2003 9(2): 255-258)

Avaliação da deformabilidade eritrocitária através da ectacitometria na deficiência de ferro

Abstract Deformability allows the 7- to 8- µ m red cells to circulatethrough capillaries of 3 µ m. This phenomenon dependson cellular geometry, internal viscosity and theviscoelastic properties of the membrane. From thevarious techniques of erythrocyte deformabilityanalysis, such as micropipette aspiration, filtration andreoscopy, we chose ectacytometry. This technique usesa laminar flow viscosimetry, where erythrocyte shapechanges are continuously monitored by laser,processed by a computer and inserted in a graph forlater analysis. Ectacytometry measures the“Deformability Index”, which shows the size ofelliptocytogenesis of the erythrocyte under “shear stress”force. Iron deficiency anemia is a very frequent diseasein medical practice. It presents expressive morphologicalterations such as microcytosis, hypochromia,ovalocytosis, elliptocytosis and target cells. Erythrocytedeformability has been described in a number ofsituations such in hereditary spherocytosis, hereditaryelliptocytosis and autoimmune hemolytic anemia. Inrespect to iron deficiency anemia, reports arecontroversial. The present study evaluates erythrocytedeformability, using ectacytometry in 21 patientsdocumented as having iron deficiency before and aftertherapy with iron components. Although the anemiatreatment proved to be efficient (before Hb – 8.52 g/dL and after Hb - 12.74 g/dL), some patients persistedwith erythrocyte morphologic alterations. Resultsdemonstrate diminished erythrocyte deformability inpeople with iron deficiency anemia, when comparedwith the control group (p< 0.0007). The absence ofregularization and maintenance of a statisticaldifference after treatment (p< 0.03) in low “shear stress”Avaliacao:

Regulation of erythrocyte ghost membrane mechanical stability by chlorpromazine

Chlorpromazine (CPZ), a widely used tranquilizer, is known to induce stomatocytic shape changes in human erythrocytes. However, the effect of CPZ on membrane mechanical properties of erythrocyte membranes has not been documented. In the present study we show that CPZ induces a dose-dependent increase in mechanical stability of erythrocyte ghost membrane. Furthermore, we document that spectrin specifically binds to CPZ intercalated into inside-out vesicles depleted of all peripheral proteins. These findings imply that CPZ-induced mechanical stabilization of the erythrocyte ghost membranes may be mediated by direct binding of spectrin to the bilayer. Membrane active drugs that partition into lipid bilayer can thus induce cytoskeletal protein interactions with the membrane and modulate membrane material properties.

Early stage shape change of human erythrocytes after application of electric field pulses

Click to increase image sizeClick to decrease image sizeErythrocyte Membrane Erythrocyte Shape Electric Field Pulse Aminophospholipid Distribution Flip-FLOP Process

Early stage shape change of human erythrocytes after application of electric field pulses.

Erythrocytes which receive electric field pulses are subject to poration, fusion and shape changes due to electrodynamic forces, aminophospholipid perturbation and influences on the normal flip-flop process. The shape change characteristics of cells suspended in different media were analysed after application of rectangular electric field pulses from t=11-44 micros and from E=4-8 kV/cm. Albumin is shown to decelerate the echinocyte shape change within the first few seconds after pulse application. The addition of fluoride and vanadate accelerates the shape change due to their inhibiting influence on the aminophospholipid translocase. For both the duration of the field pulse and its field strength, there exist lower threshold values under which no early stage shape change is observable. The activation energy calculated from the dissipative influence of the electric field alone is smaller than expected, indicating the electrodynamic influence on the flip-flop process. Cell shapes were additionally analysed by contour tracing to focus on the echinocyte spicule distribution after pulse application. This image analysis revealed that, with an increase of both pulse duration and field strength, the shape change velocity and the shape change intensity increase.

Flunitrazepam partitioning into natural membranes increases surface curvature and alters cellular morphology

In recent studies, we showed that flunitrazepam (FNTZ) and other benzodiazepines interact with artificial phospholipid membranes locating at the polar head group region, inducing a membrane expansion, reducing the molecular packing and reorganising molecular dipoles. In the present paper we investigated the possibility that those phenomena could be transduced into changes in the curvature of membranes from natural origin. Hence we studied the effect of FNTZ on cellular morphology using human erythrocyte as a natural assay system. Shape changes of erythrocytes were evaluated by light microscopy and expressed as a morphological index (MI). FNTZ induced echinocytosis in a time-dependent manner with MI values significantly higher than those of control (without drug) or DMSO (vehicle) samples. Lidocaine, a local anesthetic known to induce stomatocytosis by incorporating in the inner monolayer, counterbalanced the concentration-dependent FNTZ crenating effects. FNTZ induced protective effects, compared with control and DMSO, against time-dependent hemolysis. Hypotonic-induced hemolysis, was also lowered by FNTZ in a concentration-dependent manner. Both antihemolytic effects suggested a drug-induced membrane expansion allowing a greater increase in cell volume before lysis. In such a complex system like a cell, curvature changes triggered by drug partitioning towards the plasma membrane, might be an indirect effect exerted through modifications of ionic-gradients or by affecting cytoskeleton–membrane linkage. In spite of that, the curvature changes can be interpreted as a mechanism suitable to relieve the tension generated initially by drug incorporation into the bilayer and may be the resultant of the dynamic interactions of many molecular fluxes leading to satisfy the spontaneous membrane curvature.

HgCl 2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers

The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl 2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl 2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.