Measurement Of Red Cell Velocity Research Articles

In vivo and in vitro measurements of red cell velocity under epifluorescence microscopy

Studies of blood flow in mesentery, cremaster muscle, and small bore glass tubes were performed to obtain a relationship between mean velocity ( V mean) and red cell velocity using the two-slit method under epifluorescence ( V epi) and transillumination ( V trans) microscopy. The velocities V epi and V trans obtained in vivo for 47 measurements in arterioles and venules (12- to 51-μm internal diameter) were linearly related by V epi = 0.83 V trans + 0.074, and the ratio V epi V trans decreased gradually with increasing vessel diameter ( P ⩽ 0.05). In vitro studies in tapered glass tubes (diameter 30–70 μm) were conducted for feed hematocrits ( H F) from 10 to 40%. Under transillumination, V trans V mean was nearly constant with an average of 1.56 ± 0.16 (SD) for all hematocrits and diameters. The velocity ratio, V epi V mean , however, decreased with H F from 1.8 to 0.8 as H F was increased from 10 to 40%. Theoretical considerations suggest that the variations of V epi V mean with tube hematocrit and diameter might result from attenuation of the excitation light by absorption and scattering by red cells, and also due to a finite depth of field of the microscope objective.

Evaluation of gingival microcirculation by a laser-doppler flowmeter: Preliminary results

Up to now, only qualitative studies have been performed on gingival microcirculation whereas quantitative methods have been used in animal experiments, by the injection of microspheres for instance. The authors had the opportunity of using a Laser-Doppler Flowmeter (LDF) which had already served to explore the cutaneous microvascular blood flow. At first, it was necessary to modify an occlusal splint for good immobilisation of the probe on the gingival mucosa. Then they made a study on twenty healthy students with measurements of red cell velocity in superficial maxillary gingival mucosa. Each subject was tested twice at an interval of one-month. A statistical analysis shows very good reproducibility of the tests, especially for the pulsative velocity, despite a rather high variability between the subjects. The haemodynamic parameters (heart rate, systolic or diastolic blood pressure) do not seem to have a significant influence on the values obtained. The LDF is a very good instrument for the evaluation of gingival microvascular flow, as has been demonstrated for cutaneous microcirculation. A wide range of applications can be considered for this sensitive and harmless technique.

Microcirculatory changes in livers of mice infected with murine hepatitis virus. Evidence from microcorrosion casts and measurements of red cell velocity

Hepatitis has been considered, classically, as a diffuse hepatocellular necrosis, and little attention has been paid to the relationship of lesions to the microvasculature. In livers of mice ( Balbc J ) infected with murine hepatitis virus (MHV-3), microcorrosion casts showed spherical cavities where casting compound was unable to fill sinusoids. At 48 hr postinfection such “lesions” had a mean diameter of 83 μm ± 26 (SD) and their number/mm 2 (at the surface of casts) was 0.95 ± 1.3. Blind-ended sinusoids formed a distinct boundary between perfused and nonperfused areas, and concave impressions at their ends indicated cells blocking the lumen. In vivo microscopy of transilluminated livers in infected mice showed localized rounded areas without flow, corresponding to lesions seen in casts. RBC velocity measurements in sinusoids adjacent to lesions demonstrated that velocities fall from normal values to zero over a narrow border zone. Beginning with the most proximal sinusoid with visible flow and moving outward from the lesion to the second and third sinusoids, mean RBC velocities (μm/sec, ± SD) were 17.4 ± 6.7, 33.9 ± 8.7, 66.6 ± 27.3, respectively; this last value was not significantly different from velocities in normal liver (69.2 ± 30.6). Transmission electron microscopy of livers of infected mice confirmed the presence of sinusoidal lumens blocked by protruding lining cells, RBCs, platelets, swollen hepatocytes, and cellular debris. This study demonstrates that the lesions are focal in origin, microvascular blockage leading to gradually increasing necrosis in all directions.

Evidence for increased perfusion heterogeneity in skeletal muscle during reduced flow

Recently, it has been demonstrated ( D. Cousineau, C. P. Rose, D. Lamoureux, and C. A. Goresky, 1983, Cir. Res., 53, 719–730; K. Tyml, 1986, Microvasc. Res., 32, 84–98) that heterogeneity of microvascular flow depends on tissue metabolism. The objective of this study was to examine the possibility that, independent of metabolism, heterogeneity is also a function of flow. Using an intravital video-microscopic approach, we evaluated heterogeneity in the frog sartorius muscle at different flow rates while maintaining the muscle in the same exercised state. The flow was altered via partial aortal clamping. Exercised state was achieved by direct electrical stimulation. Heterogeneity of flow was evaluated in terms of the coefficient of variation ( CV = SD mean ) computed from simultaneous measurements of red cell velocities in a capillary network. In addition to velocity analysis, the number of perfused capillaries crossing a 1-mm test line on the video monitor was counted. Among 10 networks from eight muscles, the overall preocclusion hyperemic velocity and CV were 0.42 ± 0.15 SD mm/sec and 40 ± 12%, respectively. The overall capillary count was 16.4 ± 3.0 cap/mm. In all networks, increasing clamping reduced the mean hyperemic velocity and increased CV. For reductions to less than 50% of the preocclusion velocity, CV increased significantly ( P < 0.05), up to 88%. In 6 networks only, increasing clamping reduced capillary count, down to 10.2 cap/mm. This reduction was due to flow stoppages in capillaries situated randomly throughout the network. The data demonstrate for the first time that, for the same exercised state, heterogeneity of velocity depends on the mean velocity. This dependence is partially linked to the number of perfused vessels. When the present CV data were compared with our previously obtained resting state data ( Tyml, 1986), it became evident that, at numerically similar low mean velocity, the resting state heterogeneity can be twice as large as the exercised state heterogeneity. The present study supports the view that, in addition to the metabolic component operating at the arteriolar level, a flow-dependent hemodynamic component operating at the capillary level plays a major role in the determination of flow heterogeneity in the microvasculature.

Tube flow of human blood at near zero shear.

Pressure-velocity relations were obtained in vertical and horizontal glass tubes (I.D. 26 to 83 micron) perfused with normal human blood at feed hematocrits between 0.25 and 0.65. Perfusion pressures used corresponded to wall shear stresses up to 0.27 dyn cm-2. Red cell velocity measurements were made both immediately following implementation of perfusion pressure (with red cells still disaggregated) and in a steady state situation (with red cells aggregated). Analysis of the slopes of the linear relations between perfusion pressure and velocity showed apparent viscosity to decrease with the manifestation of red cell aggregation. In horizontal tubes, sedimentation and aggregation occurred simultaneously, and apparent viscosity increased due to axial asymmetry of cell concentration. Evidence for a yield shear stress (flow stagnation at positive driving pressure) was not observed.

CCD line-scan image sensor for the measurement of red cell velocity in microvessels

A device for measuring red cell velocities in transilluminated microvessels has been developed using a 256 element charge-coupled device (CCD) line scan sensor. An image of the moving red cells is focussed on the device so that it senses optical data along the flow axis. Paired sets of data, separated by a fixed time interval, are sampled by an A D converter operating at 1 MHz, and transferred to a dedicated microcomputer employing the MC68000 (Motorola Inc.) microprocessor. Velocity is computed from the detected spatial shift occurring in the time between data sets. The computed velocity is converted to an analogue voltage. Calibration is linear, the system is direction-sensitive and it can detect zero motion.

Possible role for adenosine in local regulation of absorptive hyperemia in rat intestine.

To test whether extracellular adenosine participates in the local regulation of intestinal blood flow during nutrient absorption, the serosa of the jejunum was continuously suffused with adenosine deaminase (7 micrograms protein/ml) or theophylline (10(-4) M) in Ringer's solution. Using video microscopy, blood flow was calculated in submucosal arterioles from diameter and red cell velocity measurements. After a steady-state baseline, oleic acid (20 mM) + glucose (56 mM) were added to a bile salt solution suffusing the mucosa. Baseline arteriolar diameters and blood flows were 52 +/- 2 micron and 20 +/- 2 nl/sec with the serosal suffusate containing Ringer's; these values were not significantly altered by theophylline or deaminase treatment. During suffusion of the mucosa with a nutrient solution, diameter and blood flow transiently increased and these responses were not altered by deaminase or theophylline. Thereafter, diameter and blood flow stabilized at lower values for the duration of absorption. Diameter and blood flow were increased to 111 +/- 1% and 134 +/- 5% of control during absorption with Ringer's; the corresponding values were significantly lower with deaminase or theophylline. After absorption, diameter and blood flow stabilized near baseline with Ringer's within 7-12 minutes; the corresponding values were significantly lower with deaminase or theophylline for at least 30 minutes. Since deaminase and theophylline produced similar effects on absorptive hyperemia, adenosine might participate with other factors in the local regulation of that response. Adenosine applied to the serosa caused dose-dependent increases in calculated blood flow with a threshold near 10(-5) M and a maximum near 10(-3) M. In contrast, even 10(-2) M adenosine in the mucosal suffusate did not increase blood flow above baseline.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo measurement of red cell velocity with epi- and transillumination

Measurement of red cell velocity with the dual-slit cross-correlation method in glass capillary tubes during transillumination indicates that the measured velocity must be divided by a correction factor of approximately 1.6 to equal the average velocity calculated from a known flow and inner diameter. Whether the same correction factor exists when red cell velocity is measured during epiillumination is questionable. Red cell velocity was measured with the dual-slit correlation method nearly simultaneously using epi- (EL) and transillumination (TL) while glass tubes (40–100 μm, i.d.) were pump perfused with whole human blood (hemotocrit 39–42%). With TL, the measured velocity is 1.58 ± 0.07 (SEM) times the calculated average velocity, whereas a factor of 2.04 ± 0.04 (SEM) was obtained with epiillumination. When intestinal arterioles with approximately the same inner diameters and flow velocities as the glass tubes were used, the ratio of velocities measured with TL to EL was 1.21 ± 0.02 (SEM) as compared to 1.31 ± 0.09 (SEM) for glass tubes using TL and EL of the tube at the same pump flow. This similarity of TL to EL velocity ratios for glass tubes and microvessels may be fortuitous or indicate that comparable flow properties and measurement conditions exist for in vitro and in vivo situations. The major finding of the study is, however, that different velocity correction factors exist for EL and TL measurements when the dual-slit correlation method is used to estimate red cell velocities in tubes of an internal diameter of 40–100 μm at normal hematocrits.

Direct measurement of microvessel hematocrit, red cell flux, velocity, and transit time.

A method is presented for the in vivo study of red cell flow dynamics. The method permits direct measurement of the red cell volume fraction in microvessel blood without resort to in vitro calibration curves. Furthermore, the method does not require extensive mathematical manipulation and can be applied to any microvascular network in any tissue. The method also enables direct measurement of red cell velocity, flux, and capillary transit time. Fluorescently labeled erythrocytes in tracer quantities, but known concentrations, are used as indicators of the behavior of the total cell population. Erythrocyte transit time across vascular networks and erythrocyte velocity are determined directly by following the behavior of the labeled cells. Hematocrit and red cell flux are measured by standard microcirculatory methods using labeled cells instead of the total cell population. Data are then converted to absolute values from the measured fraction of labeled cells. The method is thus absolutely dependent on the labeled cells being rheologically normal, and the conditions under which this requirement is satisfied are defined. Microvascular data obtained by the use of this method are presented for hamster cheek pouch and cremaster muscle.

Measurement of red cell velocity with a two-slit technique and cross-correlation: Use of reflected light, and either regulated dc or unregulated ac power supplies

The data illustrate red cell velocity values obtained from microvessels of brain and muscle by utilizing a two-slit technique and cross-correlator in settings employing reflected light. Similarities are shown between these data and data obtained or reported from transilluminated vessels. These similarities include relative independence of apparent centerline velocities on the plane of focus, and relatively low ratios of velocities at the center to velocities near the wall. It is shown, in addition, that accurate velocity measurements can be made utilizing a nonregulated ac power supply, although it cannot be stated that such measurements could be obtained in circumstances other than those described.

A method for on-line measurements of red cell velocity in microvessels using computerized frame-by-frame analysis of television images

We have used a television signal as the input to a digital computer system to measure red cell velocity in capillaries in a sartorius muscle in frogs. In principle, the velocity was measured in terms of the displacement of red cells and plasma gaps along the capillary (represented here by the shift of an optical density waveform) that occurred in a fixed time interval. Instead of using the conventional spatial cross-correlation of the waveforms, the displacement was determined by means of the absolute differences between the waveforms. The quality of the velocity measurement depended on the optical contrast between the cells and plasma gaps and an index of this quality was determined. The velocity in the capillaries of the frog muscle did not exceed 0.7 mm/sec, but it was demonstrated that, in theory, the velocity could be measured over a range of 0–21 mm/sec. This method is simple, allows further computer analysis of the velocity data and is very useful for studies of the microcirculation.

The distribution of blood rheological parameters in the microvasculature of cat mesentery.

In vivo studies of the rheological behavior of blood in the microcirculation were conducted by direct in situ measurements in cat mesentery. Upstream to downstream pressure drops were measured in unbranched arterioles, capillaries, and venules, with diameters from 7 to 58 micrometer. Simultaneous measurements of red cell velocity and vessel geometry facilitated computation of bulk velocity, pressure gradient, apparent viscosity, wall shear stress, and resistance. Arteriovenous distributions of these parameters revealed the following. Maximum pressure gradient (0.015 cm H20/micrometer) occurs in the true capillaries (7 micrometer in diameter); intravascular wall shear stress averaged 47.1 dynes/cm2 in arterioles and 29.0 dynes/cm2 in venules. Extreme values as great as 200 dynes/cm2 were observed in a few shunting arterioles. Apparent viscosity averaged 3.59 cP in arterioles, 5.15 cP in venules, and 4.22 cP overall. Intravascular resistance per unit length of microvessel varied with luminal diameter as a power law function with exponents of -4.04 for arterioles, -3.94 for venules, and -3.99 for all vessels combined. This apparent maintenance of Poiseuille's law is attributed to the opposing processes of hematocrit reduction and decreasing shear rate as blood is dispersed in successive arteriolar segments, and the converse action of these processes in the venous confluences which lessen the extent of network variations in apparent viscosity. Reductions in bulk velocity from the normal flow state to below 0.5 mm/sec resulted in increases in apparent viscosity by a factor of 2 to 10, which are attributed primarily to obstruction of the lumen by leukocyte-endothelium adhesion.

Reactive hyperemia in arterioles and capillaries of frog skeletal muscle following microocclusion.

We studied the localization of blood flow control in skeletal muscle by short-term microocclusions (30-60 seconds) of capillaries and arterioles of the pectoralis muscle in anesthetized frogs ( Rana pipiens ). The muscle was surgically exposed to permit transillumination and measurement of red cell velocity in the microvessels, but innervation and blood supply were kept intact. About one-third of the arterioles showed postocclusion hyperemia. In some muscles every arteriole showed hyperemia following occlusion, but in others none responded, presumably because of preparatory trauma. The average duration of hyperemia after a 1-minute occlusion was 74 ± 45 (SD) seconds. We also compared the effectiveness of arteriolar and capillary occlusions in producing reactive hyperemia in capillaries. Peak capillary blood flow after occlusion of the supply arteriole was 233% above control, and flow debt repayment was 278%. After occlusion of several capillaries, peak capillary blood flow was 67% above control, and flow debt repayment was 74%. In an individual capillary, peak blood flow after occlusion of that capillary was 15% above control, and flow debt repayment was 13%. In a majority of instances there was no discernible reactive hyperemia with single capillary occlusion. The results do not support the concept that flow in individual capillaries is regulated in accordance with each capillary's metabolic environment. Rather, flow in a capillary appears to depend on the metabolic environment of the arteriole supplying that capillary.