Erythrocyte aggregation and neutrophil function in an aging population.

There are limited investigations which have examined the relationship between neutrophil activation and erythrocyte aggregation in older persons. The purpose of the present study was to investigate the relationship between neutrophil activation and erythrocyte aggregation (EA) in an aging population. Twenty-eight male and female subjects were allocated into one of four groups with 7 participants in each group (group 1, 20-29 years; group 2, 30-39 years; group 3, 40-49 years; group 4, 50-59 years). EA was determined using the Myrenne aggregometer. Neutrophil function (respiratory burst and phagocytic activity) was assessed using flow cytometry. EA was found to increase with age. An ANOVA showed a significant (p < 0.05) increase for EA in autologous plasma in group 4 compared to groups 1 and 2. An ANOVA and Pearson's correlation showed that phagocytic activity decreased with age. Furthermore, a positive correlation between stimulated phagocytic activity and erythrocyte aggregability at low shear in 3% dextran-70 solution was observed. The current investigation suggests a decrease in neutrophil phagocytic activity with age and EA was increased with age. Additionally, the current study is novel as it suggests a possible relationship between neutrophil phagocytic activity and erythrocyte aggregability.

Mechanical stimulation of nitric oxide synthesizing mechanisms in erythrocytes.

It has been previously demonstrated that red blood cells (RBC) possess functional nitric oxide (NO) synthesizing mechanisms. RBC are also equipped with variety of intracellular control mechanisms, and respond to mechanical forces and to various biological stimuli by increased release of ATP. Nitric oxide has also been demonstrated to be released from RBC under certain circumstances, and it has been hypothesized that NO synthase (NOS), which is located in both the RBC membrane and cytoplasm, might be activated by mechanical factors. The present study aimed at investigating NOS activation and NO export induced by mechanical stress applied to RBC in suspension. Heparinized venous blood samples were obtained from healthy, adult volunteers and their hematocrit adjusted to 0.4 l/l. The RBC suspensions were equilibrated at room temperature (22+/-2 degrees C) with either room air or made hypoxic (36 mmHg, approximately 70% saturation) using moisturized 100% nitrogen. The samples were then continuously pumped through a glass tube (diameter = 0.06 cm; length = 33 cm) for 30 min using a dual syringe pump to maintain a wall shear stress of 0.5-2 Pa with NO concentrations in the RBC suspensions measured electrochemically. NO concentration significantly increased under the influence of 2 Pa in hypoxic RBC suspensions: 105.0+/-14.2 nM to 127.1+/-12.0 nM as the peak value at 20 min of perfusion. No increase was observed at lower levels of shear stress. Plasma nitrite/nitrate concentrations were measured in samples obtained at five minute intervals. Application of fluid shear stress to hypoxic RBC suspensions resulted in a significant, time-dependent increase of plasma nitrite/nitrate levels, reaching to 14.7+/-1.5 microM from a control value of 11.2+/-1.3 microM. The presence of the non-specific NOS inhibitor L-NAME (10(-3) M) prevented this increment. Additionally, both eNOS and serine 1177 phosphorylated eNOS immuno-fluorescence staining in RBC cytoplasm were shown to increase in response to applied shear stress. Our results support the hypothesis that RBC NO synthase is activated and that export of NO from RBC is enhanced by mechanical stress.

Inter-species differences in hematocrit to blood viscosity ratio.

Hematocrit (Hct) is the major determinant of whole blood viscosity and of its oxygen binding capacity: with increasing Hct, viscosity increases exponentially and oxygen capacity increases linearly. Thus, the theoretical oxygen transport potential of blood, as indexed by the ratio of Hct to viscosity (Hct/viscosity), generally yields a curve concave to the Hct axis with a maximum at an "optimal hematocrit" value. This study analyzed relations between Hct, blood viscosity and shear rate for rats and dogs to explore whether different optima exist for Hct or Hct/viscosity. Our results reveal differences depending on both shear rate and species: at equal Hct, rats had higher blood viscosity and thus lower Hct/viscosity levels. Optimum values for Hct/viscosity were markedly different between the two species at shear rates of 90 and 200 s-1. Conversely, Hct/viscosity data at 10 s-1 did not exhibit an optimum but rather a linear decrease of the ratio with increasing hematocrit. Relations between Hct and blood viscosity thus differ among animal species. Inasmuch as animal studies are often utilized as an aid to understanding hemorheological aspects of clinical conditions and/or therapy, evaluating Hct/viscosity ratios may be a useful supplementary tool for research focused on various physiological and patho-physiological processes.

The effects of renal ischemia-reperfusion on hemorheological factors: preventive role of allopurinol.

Changes in hemorheological parameters were studied in dogs following unilateral renal artery clamping (45-minute ischemia then reperfusion), with and without preoperative administration of allopurinol. Sham-operated animals were also evaluated. Blood samples were collected preoperatively, at beginning and at 30, 60 and 120 minutes of reperfusion, then on the 1st, 3rd, 5th and 7th days. Filtration properties of erythrocytes (relative cell transit time, RCTT), whole blood and plasma viscosity (WBV, PV), fibrinogen level and hematology parameter were determined. RCTT significantly increased for both ischemic groups at 30 minutes of reperfusion, and remained elevated on the 1st and 2nd postoperative days; these changes were abolished by allopurinol pretreatment. WBV and hematocrit increased on the 1st day, and PV and fibrinogen level showed elevation on 1st-5th postoperative days. We thus conclude that decreases of RBC deformability (i.e., higher RCTT) were characteristic and specific on early postoperative days after renal ischemia-reperfusion and that these alterations were prevented by pre-ischemia administration of allopurinol.

RBC aggregation: laboratory data and models.

The reversible aggregation of red blood cells (RBC) into linear and three-dimensional structures continues to be of basic science and clinical interest: RBC aggregation affects low shear blood viscosity and microvascular flow dynamics, and can be markedly enhanced in several clinical states. Until fairly recently, most research efforts were focused on relations between suspending medium composition (i.e., protein levels, polymer type and concentration) and aggregate formation. However, there is now an increasing amount of experimental evidence indicating that RBC cellular properties can markedly affect aggregation, with the term "RBC aggregability" coined to describe the cell's intrinsic tendency to aggregate. Variations of aggregability can be large, with some changes of aggregation substantially greater than those resulting from pathologic states. The present review provides a brief overview of this topic, and includes such areas as donor-to-donor variations, polymer-plasma correlations, effects of RBC age, effects of enzymatic treatment, and current developments related to the mechanisms involved in RBC aggregation.

Hemorheological parameters as determinants of myocardial tissue hematocrit values.

It is well known that the hematocrit in microvessels with diameters smaller than 1000 microm is lower than either venous or arterial hematocrit, thereby resulting in significantly lower mean hematocrit values for vessels perfusing a given tissue (i.e., lower tissue hematocrit). The mechanisms that underlie this reduction of microvascular hematocrit include axial migration, plasma skimming and the Fahraeus Effect. It has been previously demonstrated in rats that a linear hematocrit gradient normally exists through the thickness of the left ventricular myocardium, and that this gradient is sensitive to alterations of the rheological properties of the circulating blood. The gradient is abolished if the RBC in the perfusate are rigid; fibrinogen infusions, and thus increases of both plasma viscosity and RBC aggregation, also affect this gradient. In a new series of studies, it has been observed that enhanced RBC aggregation affects the myocardial hematocrit gradient regardless of alterations of plasma viscosity. Although the exact mechanisms responsible for the myocardial hematocrit gradient, as well as its physiological significance, are not yet clearly known, it is possible to speculate that alterations in local hematocrit could adversely affect myocardial perfusion and function.

L-carnitine deficiency and red blood cell mechanical impairment in beta-thalassemia major.

L-carnitine is an essential element of intermediary metabolism and also was shown to be effective in maintaining normal red blood cell (RBC) function. This study aimed at investigating plasma free L-carnitine concentrations and effectiveness of L-carnitine supplementation in protecting deterioration of RBC properties in beta-thalassemia major patients. Plasma free L-carnitine concentrations were determined in the blood samples obtained before their regular transfusion (about one month after the previous transfusion). Each patient received 100 mg/kg/day oral L-carnitine supplementation. RBC deformability, lipid peroxidation and intracellular free calcium concentrations were investigated before and after this treatment. Plasma free L-carnitine levels and RBC deformability before the treatment were found to be lower whereas lipid peroxidation and intracellular calcium concentration in RBC were higher compared to those of the control subjects before the L-carnitine treatment. After one month supplementation of L-carnitine lipid peroxidation and intracellular calcium concentrations were found to be decreased and RBC deformability was improved, accompanying the significantly increased plasma L-carnitine concentrations. These results suggest that L-carnitine can be used as a supplement in beta-thalassemic patients, to prevent RBC deterioration.

Activated polymorphonuclear leukocytes affect red blood cell aggregability.

Activated leukocytes can affect adjacent cells by generating oxygen free radicals and secreting proteolytic enzymes, and red blood cells (RBC) exposed to such agents should be susceptible to their effects. This study was thus designed to investigate the effects of activated polymorphonuclear leukocytes (PMN) on RBC aggregability (i.e., on intrinsic RBC aggregation characteristics). PMN were isolated from human blood by density separation and suspended in glucose-enriched buffer with RBC isolated from the same blood sample (RBC/PMN ratio of 150:1). PMN were then activated in this suspension by adding 1 ng/mL tumor necrosis factor alpha (TNF-alpha) and 10(-7) M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or fMLP alone. After incubation for 2 h at 37 degrees C, RBC aggregation behavior in autologous plasma was assessed; RBC deformability and partition coefficients were also measured. RBC aggregation was significantly increased after incubation and deformability and partitioning were decreased; these effects were prevented by phenylmethylsulfonyl fluoride (1 mM) or superoxide dismutase (20 microg/mL) plus catalase (40 microg/mL). TNF-alpha and fMLP alone had no effect on RBC aggregation if PMN were not present. Activated PMN can thus markedly affect RBC aggregability, apparently via both proteolytic enzymes and oxygen free radicals; enhanced aggregation seen in clinical states associated with PMN activation or observed during in vivo RBC aging may also involve such PMN-RBC interactions.

Effect of superoxide anions on red blood cell rheologic properties.

The human red blood cell (RBC) is known to be susceptible to oxidant damage, with both structural and functional properties altered consequent to oxidant attack. Such oxidant-related alterations may lead to changes of RBC rheologic behavior (i.e., deformability, aggregability). Two different models of oxidant stress were used in this study to generate superoxide anions either internal or external to the RBC. Our results indicate that generation of superoxide within the RBC by phenazine methosulfate decreases RBC deformability without effects on cell aggregation. Conversely, superoxide generated externally by the xanthine oxidase-hypoxanthine system primarily affects RBC aggregability: the shear rate necessary to disaggregate RBC was markedly increased while the extent of aggregation decreased slightly. Increased disaggregation shear rate (i.e., greater aggregate strength) as a result of superoxide radical damage may adversely affect the dynamics of blood flow in low-shear portions of the circulation, and may also play a role in the no-reflow phenomena encountered after ischemia-reperfusion.

Effects of swimming exercise on red blood cell rheology in trained and untrained rats.

Red blood cell (RBC) mechanical properties were investigated after swimming exercise in trained and untrained rats. A group of rats was trained for 6 wk (60 min swimming, daily), and another group was kept sedentary. Blood samples were obtained either within 5 min or 24 h after 60 min swimming in both groups. In the untrained rats, the RBC aggregation index decreased to 2.60 +/- 0.4 immediately after exercise from a control value of 6.73 +/- 0.18 (P < 0.01), whereas it increased to 13.13 +/- 0.66 after 24 h (P < 0.01). RBC transit time through 5-microm pores increased to 3.53 +/- 0.16 ms within 5 min after the exercise from a control value of 2.19 +/- 0. 07 ms (P < 0.005). A very significant enhancement (166%) in RBC lipid peroxidation was detected only after 24 h. In the trained group, the alterations in all these parameters were attenuated; there was a slight, transient impairment in RBC deformability (transit time = 2.64 +/- 0.13 ms), and lipid peroxidation was found to be unchanged. These findings suggest that training can significantly limit the hemorheological alterations related to a given bout of exercise. Whether this effect is secondary to the training-induced reduction in the degree of metabolic and/or hormonal perturbation remains to be determined.

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.

In vitro effects of thyroxine on the mechanical properties of erythrocytes.

In vitro effects of thyroxine on erythrocyte deformability and mechanical fragility were observed. Deformability of erythrocytes was improved in a dose dependent manner by thyroxine. Mechanical hemolysis was found to be lower if thyroxine was included in erythrocyte suspensions at concentrations close to the physiological levels (10(-9)M). These changes might be related to the alterations of intracellular calcium concentration, as in the erythrocyte suspensions containing 10(-9)M thyroxine, intracellular calcium concentration was found to be 30 times lower than the control suspensions which did not contain thyroxine. Thyroxine also reduced the mechanical hemolysis ratio in calcium loaded cells. These observations suggest that thyroxine might play some role in the regulation of the mechanical properties of erythrocytes which might be mediated via the effects on calcium metabolism.

Deformability of red blood cells from different species studied by resistive pulse shape analysis technique.

The Cell Transit Analyzer (CTA) is now being used widely in clinical hemorheology. Most of the data obtained by CTA are limited to human blood, although the CTA has an important potential to be used in experimental studies on animal models. However, behavior of red blood cells (RBC) from various species might be different in CTA. Eight parameters reflecting different aspects of cell passage through pores with 5 microns diameter and 15 microns length were determined or human, guinea pig, dog, rabbit, rat, mouse and sheep RBC, together with instrument precision and biological variation. These parameters have a wide range when measured in different species and correlate with cell volume. Sensitivity of these parameters to the glutaraldehyde-induced alterations in RBC deformability was not same for different laboratory mammals. The main reason for this difference seems to be related to the cell size and thus sensitivity might be significantly limited if 5 microns pore-size filters are used to test the smaller RBC. The results of this study may help in designing experimental studies on laboratory mammals using the CTA.

Cellular determinants of low-shear blood viscosity.

Low-shear viscometry is one of the methods commonly used to estimate the degree of red blood cell (RBC) aggregation in various bloods and RBC suspensions. However, it has been previously shown that alterations in RBC morphology and mechanical behavior can affect the low-shear apparent viscosity of RBC suspensions; RBC aggregation is also sensitive to these cellular factors. This study used heat treatment (48 degrees C, 5 min), glutaraldehyde (0.005-0.02%) and hydrogen peroxide (1 mM) to modify cell geometry and deformability. Red blood cell aggregation was assessed via a Myrenne Aggregometer ("M" and "MI" indexes), RBC suspension viscosity was measured using a Contraves LS-30 viscometer, and RBC shape response to fluid shear stresses (i.e., deformability) was determined by ektacytometry (LORCA system). Our results indicate that low-shear apparent viscosity and related indexes may not always reflect changes of RBC aggregation if cellular properties are altered: for situations where RBC aggregation has been only moderately affected, cellular mechanical factors may be the major determinant of low-shear viscosity. These findings thus imply that in situations which may be associated alterations of RBC geometry and/or deformability, low-shear viscometry should not be the sole measurement technique used to assess RBC aggregation.

Determination of red blood cell shape recovery time constant in a Couette system by the analysis of light reflectance and ektacytometry.

Red blood cell (RBC) shape change under shear is generally reversible, with the time course of shape recovery a function of the elastic and viscous properties of the RBC membrane. RBC shape recovery can be investigated, using several different techniques, to provide information about the membrane material properties that are not directly accessible by frequently used methods to assess RBC deformability (e.g., micropore filtration). In the present study, RBC shape recovery was studied in a Couette system after abrupt cessation of shear, either by analyzing the time course of laser light reflection or by serial measurements of elongation indexes from laser diffraction patterns. The time course of shape recovery monitored with both techniques can be described with an exponential equation. Calculated time constants for normal human RBC were 119 +/- 17 msec and 97 +/- 15 msec as measured by light reflection and ektacytometry, respectively. Treatment of RBC with glutaraldehyde resulted in dose-dependent decreases in the shape recovery time constant. Heat treatment (48 degrees C, 20 min), which is known to increase mainly the shear elastic modulus of the membrane, decreased the time constant by 65%. In contrast, wheat germ agglutinin treatment increased the shape recovery time constant by 22%, presumably by increasing membrane surface viscosity. Our results indicate that the shape recovery time constant of RBC can be measured easily and accurately by computerized light reflection analysis.

Influence of cell-specific factors on red blood cell aggregation.

The reversible aggregation of red blood cells (RBC) into linear and three-dimensional structures continues to be of basic science and clinical interest: RBC aggregation affects low shear blood viscosity and microvascular flow dynamics, and can be markedly enhanced in several clinical states. Until fairly recently, most research efforts were focused on relations between suspending medium composition (i.e., protein levels, polymer type and concentration) and aggregate formation. However, there is now an increasing amount of experimental evidence indicating that RBC cellular properties can markedly affect aggregation, with the term "RBC aggregability" coined to describe the cell's intrinsic tendency to aggregate. Variations of aggregability can be large, with some changes of aggregation substantially greater than those resulting from pathologic states. The present review provides a brief overview of this topic, and includes such areas as donor-to-donor variations, polymer-plasma correlations, effects of RBC age, effects of enzymatic treatment, and current developments related to the mechanisms involved in RBC aggregation.

Aggregation behavior and electrophoretic mobility of red blood cells in various mammalian species.

Differences of red blood cell (RBC) aggregation among various mammalian species has been previously reported for whole blood, for RBC in autologous plasma, and for washed RBC re-suspended in polymer solutions. The latter observation implies the role of cellular factors, yet comparative studies of such factors are relatively limited. The present study thus investigated RBC aggregation and RBC electrophoretic mobility (EPM) for guinea pigs, rabbits, rats, humans and horses; RBC were re-suspended in isotonic 500 kDa dextran solutions for the EPM and aggregation measurements, with aggregation studies also done in autologous plasma. Salient results included: (1) species-specific RBC aggregation in both plasma and dextran (horse > human > rat > rabbit approximately = guinea pig) with a significant correlation between aggregation in the two media; (2) similar EPM values in PBS for rat, human and horse, a lower value for guinea pig, and a markedly reduced EPM for rabbit RBC; (3) EPM values in dextran with a rank order identical to that for cells in PBS; (4) relative EPM results indicating formation of a polymer-poor, low viscosity depletion layer at the RBC surface (greatest depletion for horse RBC). EPM-aggregation correlations were evident and generally consistent with the Depletion Model for aggregation, yet did not fully explain differences between species; additional studies at various ionic strengths and with various dextran fractions thus seem warranted.

Red blood cell rheological alterations in a rat model of ischemia-reperfusion injury.

Red blood cell (RBC) deformability and aggregation characteristics were investigated in an experimental model of ischemia-reperfusion injury. Ischemia was produced in rat hind limb by occluding the femoral artery for 10 minutes, followed by reperfusion. Blood samples were obtained either following the ischemia or 15 minutes after reperfusion. RBC deformability measured by ektacytometry was found to be significantly impaired immediately after the end of ischemic period in the blood samples obtained from femoral vein of the ischemic limb, while there was no significant difference after 15 minutes of reperfusion. In contrast, RBC aggregability was found to be decreased only after the reperfusion period and this alteration was not only limited to the blood returning from the ischemic limb but was also observed in the samples obtained from non-ischemic, contralateral hind limb, indicating a systemic alteration. RBC electrophoresis studies suggested that the altered aggregability might be related to altered RBC surface properties including increased RBC surface charge density.

The role of spleen in suppressing the rheological alterations in circulating blood.

The role of spleen in maintaining the normal rheological properties of red blood cells (RBC) has been investigated by comparing the time course of RBC deformability assessed by the cell transit analyzer (CTA), after the induction of RBC mechanical alterations, in splenectomized and normal guinea pigs. After the exchange transfusions with glutaraldehyde treated (hardened) RBC, most of these cells were removed from the circulation in the splenectomized animals as well as the animals with intact spleens. However, the CTA could detect the longer existence of a small population of hardened RBC in circulation in the splenectomized animals. Measurement of RBC transit times after the onset of experimental sepsis (cecal ligature-puncture) revealed that, in the splenectomized guinea pigs RBC deformability impairment started earlier, in comparison with the animals with intact spleens. These results suggest that the spleen plays an important role in maintaining the normal rheological properties of the circulating blood, especially in the presence of pathophysiological processes affecting RBC mechanics.

Importance of measurement temperature in detecting the alterations of red blood cell aggregation and deformability studied by ektacytometry: a study on experimental sepsis in rats.

It is well known that RBC rheological parameters are affected by temperature. Usually, the measurement temperature of these parameters is kept constant throughout a given study, however it can be seen that different temperatures are used by different groups, during the measurement of a given parameter. It is assumed that the data should not be qualitatively different when measured at different temperatures, although significant quantitative differences exist. This study revealed that the selected temperature for RBC elongation index measurements by ektacytometry is important in detecting a given impairment in RBC deformability induced by experimental sepsis. RBC elongation indexes were found to be significantly different in septic and normal rats, only if measured at 37 degrees C. The differences in RBC aggregation parameters for septic and normal rats were also affected by the measurement temperature, however statistically significant differences were present in a wider range of temperatures between 25-37 degrees C. Therefore, it is strongly recommended that the measurement of RBC deformability and aggregation be performed using a controlled-temperature device, set to 37 degrees C.