H. Schmid-Schönbein

Tank Tread Motion of Red Cell Membranes in Viscometric Flow: Behavior of Intracellular and Extracellular Markers (with Film)

With a cone and plate ‘rheoscope’, red cell suspensions can be observed while subjected to shear. Interference contrast optics and high speed cinematography allow resolution of details of deformation in native, unstained red blood cells (RBC) with an optical resolution of 0.3 μm and a time resolution of 2 ms. Movement of membrane and cytoplasm of RBCs is monitored by various markers. At shear rates above 500/s, red cells in whole blood (Hct > 40%) show elongation parallel to flow direction, whereas single red cells suspended in plasma (Hct < 5%) take irregular polyhedral forms and tumble in a shear flow at the same shear rate. Single cells suspended in viscous solutions (e.g. Dextran) under shear are deformed to form flat ellipsoids. Their elongation increases asymptotically towards a maximum with increasing shear stress. Ellipsoidally deformed RBCs assume a stationary orientation in the shear field, the long axis lying in a plane through the center of the cell containing the flow direction and perpendicular to the plate of the rheoscope. The angle between the long axis and the flow direction is zero only for very small and very large elongations. The membrane shows tank tread motion, the frequency of which increases linearly with the shear rate. Shear flow is transmitted from the continuous phase into the cytoplasm of the cell and can be observed directly by cytoplasmic markers. The shear rate within the cytoplasm is approximately constant. Assuming Newtonian behavior of both the continuous phase and the intracellular hemoglobin solution, the calculated shear stresses within the cell are lower by a factor of 4 than those in the continuous phase. Moderate stomatocytes and echinocytes (I and II) are also deformed to form stationary ellipsoids, irrespective of their preparation by different agents. The shear stresses required for their elongation are of the same order of magnitude as those required for discocyte elongation. Fragmentation of RBCs in shear flow and an altered appearance of the membrane after its plastic deformation in excessive shear could be observed.

Conceptional Proposition for a Specific Microcirculatory Problem : Maternal Blood Flow in Hemochorial Multivillous Placentae as Percolation of a “Porous Medium”

Recently, it has been convincingly shown by Murphy et al. (1986) that hemoconcentration is a risk factor in severe pre-eclampsia. More specifically, the work of Heilmann et al. (1977, 1984) has shown that the non-Newtonian behavior of blood is distinctly more pronounced in patients suffering from pre-eclampsia and other forms of placental dysfunction. In these pathologic pregnancies, many of which are known to have a high uterine artery impedence to flow (Campbell et al., 1983; Fendei, 1985), there is evidence that curtailed fluidity of maternal blood might criticially limit the perfusion of the intervillous spaces (a topic recently covered in an international symposium, cf. Heilman, 1984).

Experimental calibration of a two-stage prism-grating system for measuring cell velocity

Abstract A new optical system for the measurement of the erythrocyte velocity in microvessels is extensively applied for the first time. It is based on the projection of the erythrocyte image onto two photodiodes through a prism grating. The differential signal of the photodiodes is proportional to the velocity. The system is comparable to those of M. Anliker, M. Casty, P. Friedli, R. Kubli, and H. Keller ((1977). Noninvasive measurement of blood flow. In “Cardiovascular Flow Dynamics and Measurement” (N. H. Hwang and N. Normann, eds.), pp. 43–88. Univ. Park Press, Baltimore) and D. W. Slaaf, J. P. S. M. Rood, G. J. Tangelder, and T. Arts ((1979). Microvasc. Res. 17, S173). It measures an instantaneous velocity. The system is calibrated by a velocity which is constant over the total measuring field and by a known flow rate and profile. Concerning applicability and results the device is compared to the two-slit methods, the laser-Doppler-anemometry and high-speed cinematography.

Ultrastructural Investigations on the Question of Mechanical Activation of Blood Platelets

SummaryThe present study addresses the question whether platelets are activated by mechanical stresses as they occur in pathologically accelerated blood flow. Their potential mechanoreceptive properties were tested by subjecting human platelets to defined fluid mechanical forces for periods of milliseconds. Platelet activation was assessed by quantitative morphology, revealing besides activated platelets, irreversibly ballooned, lytic platelets. However, this morphometrically documented “shear activation” of platelets can be suppressed almost completely by the addition of enzyme-substrate systems, capable of removing adenosine diphosphate from the suspending medium. This is in keeping with a recent study from our laboratory [27] showing that the behaviour of lactic dehydrogenase as marker for platelet lysis and β-thromboglobulin as release marker refuted earlier data, suggesting a direct activation of platelets by shear. It is concluded that former evidence of “shear induced platelet activation” must be interpreted as the consequence of lytic damage to a small proportion of platelets.

Standardized ultrasound as a new method to induce platelet aggregation. Evaluation, influence of lipoproteins and of glycoprotein IIb/IIIa antagonist tirofiban

Most of the published studies concerning platelet aggregation were performed with chemical stimulation procedures, however, mechanical stimulation might be a better simulation of physiological activation of platelets. In order to evaluate the influence of ultrasound on platelet aggregation in vitro, we developed an ultrasound device in a standardized set-up, and we evaluated the influence of lipoproteins and the glycoprotein IIb/IIIa inhibitor tirofiban on ultrasound induced platelet aggregation. A cylindrical shaped plastic test tube with 1 ml of platelet-rich plasma was placed in an ultrasound bath (35 kHz) for 5 s. The ultrasound energy transfer into the sample (Delta W=3.77 J) was calculated using the average temperature increase (averaged by 0.935 degrees C) of the sample. Platelet aggregation was quantified immediately after stimulation with ultrasound or adenosine diphosphate (ADP 2.1 and 4.2 microM) by the Myrenne Aggregometer PA2 at low (40 s(-1)) and afterwards at high (2500 s(-1)) shear. To evaluate the influence of lipoproteins, seven healthy male volunteers were investigated before and after a fat load (50 g fat per m(2) body surface), and 11 patients suffering from hypercholesterolemia and atherosclerotic disease before and after a single low-density lipoprotein (LDL) apheresis. Platelet aggregation after ultrasound stimulation was well correlated with platelet aggregation after ADP (r between 0.50 and 0.95). However, when exposed to high shear, the low shear-induced platelet aggregates were more stable after ultrasound stimulation compared with ADP stimulation either with or without tirofiban. After the fat load triglyceride concentration increased from 0.86+/-0.39 to 2.10+/-1.10 mmol l(-1) (P<0.05) resulting in a reduced formation of platelet aggregates after weak (ADP 2.1 microM) but not after strong (ADP 4.2 microM or ultrasound) stimuli. After a single LDL apheresis LDL cholesterol dropped from 3.99+/-0.90 to 1.06+/-0.55 mmol l(-1) (P<0.005). No changes in platelet aggregation were observed with the exception of a lower aggregation when exposed to high shear after stimulation with 2.1 microM ADP. In conclusion, we found the ultrasound stimulation of platelet-rich plasma easy to perform. The platelet aggregation after ultrasound stimulation correlated well with stimulation after ADP. While a reduction in LDL cholesterol concentration had only slight effects on platelet aggregation, an increase in triglyceride concentration resulted in a reduced formation of platelet aggregates after weak stimulation.

Analysis of network flow distribution: Computational aid to minimize experimental expenditure ☆

Several problems in microvascular research, e.g., total blood flow and red cell flux distribution, can only be solved by evaluating the flow distribution to all branches of microvessel networks. We present a method to obtain values of volumetric blood flow and red cell flux in individual networks by taking measurements in an adequately chosen number of the vessels (50-33%) in the network. Actual measurements are not mandatory in all vessels because in a major part of them computational quantification of flows and fluxes is possible. The proposed method consists of a procedure to document in matrix form the connections of a network found in a preparation. By appropriate mathematical manipulations of the matrix, a preselection of measuring sites within the network can be achieved. From the flow values in these sites the remaining vessel flows can be calculated by utilizing the law of mass conservation in all branching points and confluxes. The worst case error of the procedure can be calculated from the errors of the single measurements. The method can also be applied, whenever, for experimental reasons (e.g., poor microscopic focus, flow too rapid or too slow), certain branches do not lend themselves for measurement. A BASIC program can be made available upon request, which carries out all calculations necessary for an appropriate selection in such a short time (about 1 min) so that it can easily be performed before the start of data acquisition.

A comparative study: Perfusion of the micro- and macrocirculation as a function of the hematocrit value

The fluidity (the inverse of viscosity) of red blood cell (RBC) suspensions in vivo was estimated by means of microcirculatory measurements such as RBC flow velocity, micropressure in arterioles and venules, and vessel geometry, and by means of simultaneous macrocirculatory measurements such as volume flow and perfusion pressure in the whole organ. These in vivo data were compared to in vitro data obtained by viscometry. The experiments were performed on the isolated rat mesentery perfused with a nonaggregating and an aggregating suspension: Human RBC were either suspended in Ringers or Ficoll 400 solution. The tissue was perfused at various perfusion pressures and hematocrit values. The microcirculatory perfusion was recorded by means of microscopy using a video TV system. Five major results have been identified: First, microfluidities calculated from velocity data in capillaries of the mesenteric membrane were almost identical to the fluidities calculated from macroflow data (volume flow rates and perfusion pressure in the whole organ). Second, in vivo fluidities of both suspensions decrease with increasing hematocrit value but seem to be independent of driving pressure between 4 and 10 kPa. Third, the fluidity of the Ficoll suspensions is lower than the fluidity of the Ringers suspensions by the same ratio as the continuous phases. Fourth, the in vivo fluidities of the Ringers suspensions and the Ficoll suspensions for hematocrit values from 20 to 70% and a range of wall shear stresses from 0.6 to 1 Pa were higher than those measured in vitro. Finally, it has been quantified that an increasing number of vessels became stagnant (that is packed by red blood cells) at high hematocrit values, low perfusion pressure, and increased aggregability of RBC.

Change of Local Charge Density by Change of Local Mean Curvature in Biological Bilayer Membranes

Abstract Highly positive curved parts of biological membranes show an increase in density of charge carrying molecules, while parts with less positive or negative mean curvature show a diminution. This has been shown by different investigators by marking the charge carrying molecules and examining the membranes by electron microscopy. We here give an explanation for this finding based on electrostatics field theory. Assuming free lateral mobility of charge carrying molecules the membrane forms an equipotential surface. Under this premise the socalled charge simulation method can be used for building up computer models for the electrostatic properties of different shaped membranes. These models show the same behaviour for the charge density distribution as has been observed in the distribution of charge carrying molecules in membranes. So simple electrostatic repulsion forces may also in biological membranes be the reason for local mean curvature related changes in charge carrying molecules distribution.

Zur Untersuchung der Retinadurchblutung mittels Fernsehfluoreszenzangiographie

Die Fernsehfluoreszenzangiographie ist ein Verfahren, das sowohl die Registrierung von konventionellen Netzhautaufnahmen als auch von Fluoreszenzangiogrammen ermoglicht Die Aufzeichnung von Angiogrammen bei guter Auflosung und minimaler Beleuchtungsstarke ist inzwischen realisiert Mit Hilfe eines selbstentwikkelten Videotimers und -analysers ist es moglich, reproduzierbar Farbstoffdilutionskurven zu registrieren. Aus diesen lassen sich verschiedene, fur die Mikrozirkulation relevante, Parameter ermitteln.

A Measuring Device to Determine a Universal Parameter for the Flow Characteristics of Blood: Measurement of the Yield Shear Stress in a Branched Capillary

There has been an increasing attempt in recent years to search for alterations in hemorheological parameters in a variety of disease states, characterized by an increase in the viscosity of whole blood (Volger, 1980). Factors which influence this gross parameter include: hematocrit, plasma viscosity, extent of erythrocyte aggregation, and red cell deformability. A number of controversial methods exist to quantify these factors, but until recently it has been impossible to evaluate the total extent of a rheological alteration or to quantify the influence of a change in several parameters on the whole blood viscosity. The most commonly used measurement of whole blood viscosity — “rotational viscosimetry” — seems to be inappropriate because certain pathological states are concealed by phase separation in the measuring chamber due to sedimentation of large erythrocyte aggregates. This results in an apparent whole blood viscosity, as measured by rotational viscosimetry, much lower than the true viscosity in samples with greatly altered rheological parameters.