Viscoelasticity of human alveolar epithelial cells subjected to stretch

Alveolar epithelial cells undergo stretching during breathing and mechanical ventilation. Stretch can modify cell viscoelastic properties, which may compromise the balance of forces in the alveolar epithelium. We studied the viscoelasticity of alveolar epithelial cells (A549) subjected to equibiaxial distention with a novel experimental approach. Cells were cultured on flexible substrates and subjected to stepwise deformations of up to 17% with a device built on an inverted microscope. Simultaneously, cell storage (G') and loss (G') moduli were measured (0.1-100 Hz) with optical magnetic twisting cytometry. G' and G' increased with strain up to 64 and 30%, respectively, resulting in a decrease in G'/G' (15%). This stretch-induced response was inhibited by disruption of the actin cytoskeleton with latrunculin A. G' increased with frequency following a power law with exponent alpha = 0.197. G' increased proportionally to G' but exhibited a more marked frequency dependence at high frequencies. Stretching (14%) caused a fall in alpha (13%). At high stretching amplitudes, actual cell strain (14.4%) was lower than the applied substrate strain (17.3%), which could indicate a partial cell detachment. These data suggest that cytoskeletal prestress modulates the elastic and frictional properties of alveolar epithelial cells in a coupled manner, according to soft glassy rheology. Stretch-induced cell stiffening could compromise the balance of forces at the cell-cell and cell-matrix adhesions.     

Microwave dielectric measurements of erythrocyte suspensions.

Complex dielectric constants of human erythrocyte suspensions over a frequency range from 45 MHz to 26.5 GHz and a temperature range from 5 to 40 degrees C have been determined with the open-ended coaxial probe technique using an automated vector network analyzer (HP 8510). The spectra show two separate major dispersions (beta and gamma) and a much smaller dispersion between them. The two major dispersions are analyzed with a dispersion equation containing two Cole-Cole functions by means of a complex nonlinear least squares technique. The parameters of the equation at different temperatures have been determined. The low frequency behavior of the spectra suggests that the dielectric constant of the cell membrane increases when the temperature is above 35 degrees C. The real part of the dielectric constant at approximately 3.4 GHz remains almost constant when the temperature changes. The dispersion shifts with temperature in the manner of a thermally activated process, and the thermal activation enthalpies for the beta- and gamma-dispersions are 9.87 +/- 0.42 kcal/mol and 4.80 +/- 0.06 kcal/mol, respectively.     

Relaxation phenomena in human erythrocyte suspensions.

Previous work has shown that the application of the Joule heating temperature jump technique of Eigen and de Maeyer to an istonic suspension of human erythrocytes induced an interiorization of [3H-A1glucose and a hemolysis of the red cells (Tsong, T.Y., and E. Kingsley, J. Biol. Chem. 250:786 [1975]). The result was interpreted as due to the thermal osmosis effect. Further considerations of the various effects of the Joule heating technique indicate that the hemolysis of the red cells may also be caused by the rapid dielectric perturbation of the cell membranes. By means of turbidity measurements of the suspensions we have detected at least four relaxation times. Two of the faster ones (tau1 approximately 20 mus and tau2 approximately 5 ms) are tentatively attributed to water relaxations in the membrane structures. The other two are attributed to membrane ruptures (tlag approximately 0.1s) and the hemolysis reaction (tau3 approximately 0.5 s). Studies with the erythrocytes from different hematological disorders indicate that whereas the two slower relaxations are sensitive to the overall physical property of the red cell membranes the two faster relaxations are not. These observations are consistent with the above assignment of the relaxation processes. The apparent activation energies are, above assignment of the relaxation processes. The apparent activation energies are, respectively, 8.4, 12.0, and 11.8 kcal/mol for the tau1, tau2, and tau3 reactions. Experiments with erythrocyte ghosts indicate a single relaxation for the water permeation, and biphasic kinetics for the membrane rupture and resealing reactions. The phenomena reported here may contribute to our understanding of water transport and molecular release in cellular systems.     

Flash freezing of erythrocyte suspensions

Freezing whole blood in bulk usually results in severe cellular destruction through the action of ice crystals and osmotic effects in the freezing liquid. The potential of flash freezing blood aerosols onto a liquid nitrogen surface as a means of inhibiting cellular damage was studied in this work. Three commercial spraying devices were employed to spray-freeze either whole blood or concentrated erythrocyte suspensions, using hydroxyethyl starch (HES) as a cryoprotectant. The integrity and viability of the processed cells were assessed by measuring gross rheological properties and the extent of hemolysis. Cells were found to be susceptible to the very high shear stresses imposed by some of the spraying devices. Bulk freezing of blood, even in the presence of the cryoprotectant, resulted in complete cellular destruction. Whereas flash freezing was capable of substantially reducing the level of hemolysis to 12.6% and preserving the cellular deformability.     

Heat transport in laminar flow of erythrocyte suspensions.

Measurements of thermal conductivity were made in laminar flow of dog and turkey erythrocyte suspensions in a stainless stell tube of about 1 mm ID. These measurements were independent of the shear rate, showing that the red cell motion relative to plasma in flowing blood had no effect on the heat transfer. Measurements of thermal conductivity were further made in suspensions of polystyrene spheres of 100 mum and were found to be dependent upon the shear rate. The Graetz solution corresponding to uniform wall temperature was used for determining the value of thermal conductivity in an apparatus calibrated with tap water. The overall accuracy of the results is within 10%. A model based on the particle rotation with the entrained fluid is proposed. It is pointed out that the diffusion of platelets, red cells, and possibly plasma proteins (such as fibrinogen) will be augmented if they happen to be in the hydrodynamic field of rotating erythrocytes.     

Application of some droplet sedimentation theories to layered erythrocyte suspensions

The applicability of some existing droplet sedimentation theories to erythrocyte suspensions was investigated using glutaraldehydefixed erthrocytes from horse, canine, pig, chicken, and human. The Svensson criteria were shown to underestimate the load supportable by a density gradient. The available theories on droplet formation times could not predict each other, and the data on erythrocyte suspensions, especially at high suspension concentrations. It was finally argued that, since the behavior of erythrocytes was not controlled by the diffusion process, the erythrocyte suspensions were not expected to exhibit lasting or absolute stability even when the particle load was small.     

Application of some droplet sedimentation theories to layered erythrocyte suspensions

The applicability of some existing droplet sedimentation theories to erythrocyte suspensions was investigated using glutaraldehyde-fixed erythrocytes from horse, canine, pig, chicken, and human. The Svensson criteria were shown to underestimate the load supportable by a density gradient. The available theories on droplet formation times could not predict each other, and the data on erythrocyte suspensions, especially at high suspension concentrations. It was finally argued that, since the behavior of erythrocytes was not controlled by the diffusion process, the erythrocyte suspensions were not expected to exhibit lasting or absolute stability even when the particle load was small.     

Measurements of viscosity of synthetic erythrocyte suspensions

Measurements were made of the viscosity of suspensions of synthetic erythrocytes composed of hemoglobin solutions encapsulated in liposomes, as a function of shear rate, temperature, suspension concentration, lipid membrane composition, and the viscosity of the suspending medium. It was found that the viscous behavior of the synthetic erythrocyte suspensions was non-Newtonian and nearly the same as that of suspensions of natural erythrocytes prepared similarly, with the major difference being that synthetic erythrocyte suspensions are somewhat more viscous. Suspensions of Fluosol FC-43 prepared similarly were found to be essentially Newtonian fluids, and substantially different and more viscous than either erythrocyte suspension. The higher viscosity of synthetic erythrocyte suspensions probably accounts for the ability of these suspensions to maintain normal systemic vascular resistance in transfusion experiments, in spite of the fact that synthetic erythrocytes are smaller than natural erythrocytes.     

Force relaxation and permanent deformation of erythrocyte membrane.

Force relaxation and permanent deformation processes in erythrocyte membrane were investigated with two techniques: micropipette aspiration of a portion of a flaccid cell, and extension of a whole cell between two micropipettes. In both experiments, at surface extension ratios less than 3:1, the extent of residual membrane deformation is negligible when the time of extension is less than several minutes. However, extensions maintained longer result in significant force relaxation and permanent deformation. The magnitude of the permanent deformation is proportional to the total time period of extension and the level of the applied force. Based on these observations, a nonlinear constitutive relation for surface deformation is postulated that serially couples a hyperelastic membrane component to a linear viscous process. In contrast with the viscous dissipation of energy as heat that occurs in rapid extension of a viscoelastic solid, or in plastic flow of a material above yield, the viscous process in this case represents dissipation produced by permanent molecular reorganization through relaxation of structural membrane components. Data from these experiments determine a characteristic time constant for force relaxation, tau, which is the ratio of a surface viscosity, eta to the elastic shear modulus, mu. Because it was found that the concentration of albumin in the cell suspension strongly mediates the rate of force relaxation, values for tau of 10.1, 40.0, 62.8, and 120.7 min are measured at albumin concentrations of 0.0, 0.01, 0.1, and 1.% by weight in grams, respectively. The surface viscosity, eta, is calculated from the product of tau and mu. For albumin concentrations of 0.0, 0.01, 0.1, and 1% by weight in grams, eta is equal to 3.6, 14.8, 25.6, and 51.9 dyn s/cm, respectively.     

The activity of erythrocyte enzymes in rats subjected to running exercises

The studies were carried out on male Wistar rats subjected to running within an electric rotating drum. The animals were divided into four experimental groups, differing one from another as to the duration of training. Each training session lasted 30 days. In the first group the daily run lasted 3 min, in the second group 5 min; in the third group, a 1 min run on the first day, and one min longer on each successive day; in the fourth group a 2 min run on the first day and for two min longer on each successive day. The determinations made prior to and after training included the peripheral blood erythrocyte (Er) and reticulocyte (Ret.) count, the hemoglobin concentration (Hb) and packed cell volume (PCV) and, determined by spectrophotometric methods, the activity of pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR). Training induced an improvement of all enzymatic activities. The heavier the physical exertion, the more intensive was the enzymatic activity of red blood cells, due to the intensification of bone marrow erythropoetic activity under physical exertion and the appearance of young red cells in peripheral blood. All the experimental groups revealed a drop in erythrocyte count (Er), hemoglobin concentration (Hb), and hematocrit values (PCV), as well as an increase in the reticulocytes count (Ret) and in the activity of all the enzymes investigated. In the fourth group anemia was detected: prolonged endurance training decreased the RBC by 24.2%, Hb by 31.1%, PCV by 26.2% and increased the reticulocyte count by 881.6%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusion of fluorescein-labelled molecules in suspensions of erythrocyte ghosts

The diffusion of fluorescein isothiocyanate-labelled dextran molecules in suspensions of centrifugally, tightly packed, erythrocyte ghosts was measured by fluorescence recovery after photobleaching. In comparison with diffusion in aqueous solution, the diffusion coefficients for probe molecules of varying size were about two orders of magnitude smaller. It was established that the dextran molecules remained in the space between the ghosts. Since crosslinking membrane surface carbohydrates with antibodies further inhibits diffusion, it is assumed that interactions between surface carbohydrates and the probe molecules are the cause of slow diffusion. Two alternative models are discussed.     

Factors affecting yield and survival of cells when suspensions are subjected to centrifugation

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.     

Microscopic investigation of erythrocyte deformation dynamics

The understanding of erythrocyte deformation under conditions of high shear stress and short exposure time is central to the study of hemorheology and hemolysis within prosthetic blood contacting devices. A combined computational and experimental microscopic study was conducted to investigate the erythrocyte deformation and its relation to transient stress fields. A microfluidic channel system with small channels fabricated using polydimethylsiloxane on the order of 100 mum was designed to generate transient stress fields through which the erythrocytes were forced to flow. The shear stress fields were analyzed by three-dimensional computational fluid dynamics. Microscopic images of deforming erythrocytes were experimentally recorded to obtain the changes in cell morphology over a wide range of fluid dynamic stresses. The erythrocyte elongation index (EI) increased from 0 to 0.54 with increasing shear stress up to 123 Pa. In this shear stress range, erythrocytes behaved like fluid droplets, and deformed and flowed following the surrounding fluid. Cells exposed to shear stress beyond 123 Pa (up to 5170 Pa) did not exhibit additional elongation beyond EI=0.54. Two-stage deformation of erythrocytes in response to shear stress was observed: an initial linear elongation with increasing shear stress and a plateau beyond a critical shear stress.     

Micro-macro link in rheology of erythrocyte and vesicle suspensions

We report on the rheology of dilute suspensions of red blood cells (RBC) and vesicles. The viscosity of RBC suspensions reveals a previously unknown signature: it exhibits a pronounced minimum when the viscosity of the ambient medium is close to the value at which the transition from tank-treading to tumbling occurs. This bifurcation is triggered by varying the viscosity of the ambient fluid. It is found that the intrinsic viscosity of the suspension varies by about a factor of 4 in the explored parameter range. Surprisingly, this significant change of the intrinsic viscosity is revealed even at low hematocrit (5%). We suggest that this finding may be used to detect blood flow disorders linked to pathologies that affect RBC shape and mechanical properties. This opens future perspectives on setting up new diagnostic tools, with great efficiency even at very low hematocrit. Investigations are also performed on giant vesicle suspensions, and compared to RBCs.     

Inheritance of low erythrocyte catechol-o-methyltransferase activity in man.

Catechol-O-methyltransferase activity was measured in blood obtained from 373 randomly selected subjects aged 16-18, 262 consecutive adult blood donors, and 201 first-degree relatives of subjects with RBC COMT activity of less than 8 U. The distribution of RBC COMT activity in a randoly selected populations was apparently bimodal with a nadir at approximately 8 U. Of a randomly selected population, 23% had low RBC COMT activity (less than 8 U), Because of previous reports of a significant sibling-sibling correlation of RBC COMT activity and because of the presence of a subgroup of subjects with low enzyme activity, RBC COMT activity was measured in blood from first-degree relatives of probands with low erythrocyte enzyme activity in 48 families. The results of segregation analyses of the data were compatible with autosomal recessive inheritence of an allele for low RBC COMT activity. RBC COMT in blood samples from siblings of probands inthese families also showed an apparent biomodal distribution.     

Simulations of the erythrocyte cytoskeleton at large deformation. I. Microscopic models.

Three variations of a polymer chain model for the human erythrocyte cytoskeleton are used in large deformation simulations of microscopic membrane patches. Each model satisfies an experimental observation that the contour length of the spectrin tetramers making up the erythrocyte cytoskeleton is roughly square root of 7 times the end-to-end distance of the tetramer in vivo. Up to modest stress, each brushy cytoskeletal network behaves, consistently, like a low-temperature, planar network of Hookean springs, with a model-dependent effective spring constant, keff, in the range of 20-40 kBT/s(o)2, where T is the temperature and s(o) is the force-free spring length. However, several features observed at large deformation distinguish these models from spring networks: 1) Network dimensions do not expand without bound in approaching a critical isotropic tension (square root of 3 keff) that is a characteristic limit of Hookean spring nets. 2) In surface compression, steric interactions among the chain elements prevent a network collapse that is otherwise observed in compression of planar triangulated networks of springs. 3) Under uniaxial surface tension, isotropy of the network disappears only as the network is stretched by more than 50% of its equilibrium dimensions. Also found are definitively non-Hookean regimes in the stress dependence of the elastic moduli. Lastly, determinations of elastic moduli from both fluctuations and stress/strain relations prove to be consistent, implying that consistency should be expected among experimental determinations of these quantities.     

X-ray scattering studies of heavy atom bound erythrocyte membrane suspensions

We have observed a periodic intensity variation of scattered x-rays from uranyl labeled erythrocyte membranes. Using unique x-ray scattering methods, we have made these measurements from membrane suspensions in which the vesicles appear, by phase contrast microscopy, to be normal in shape. The periodic intensity variation is not present for membranes labeled on one side only. The frequency of the variation permits calculation of membrane width, which we find to be 55Å.