Effect of Hematocrit

Whole blood viscosity is dependent on the number (and volume) of erythrocytes in the blood, and is thus linearly related to hematocrit (Chien et al., 1975). The impact of hematocrit on blood viscosity is much higher at low shear rates (veins for instance) than at high shear rate (arteries for instance) (Cokelet and Meiselman, 2007). At high shear rate, it is estimated that a rise of hematocrit of one unit would cause an increase of blood viscosity of 4% (if RBC rheological properties remain the same).

Plasma Viscosity

Plasma is a newtonian fluid, which means that its viscosity does not vary with shear rate. The viscosity of plasma is dependent on the concentration of plasma proteins, such as fibrinogen, α1-globulins, α2-globulins, β-globulins, and γ-globulins (Connes et al., 2008). Any elevation in the concentration of these proteins can cause plasma, and thus whole blood, viscosity to increase (Kesmarky et al., 2008). Normal plasma at 37 degrees Celsius has a viscosity of around 1.2–1.3 cP, but these values may be higher in various inflammatory, metabolic, or cardiovascular diseases (Kesmarky et al., 2008). Furthermore, increased plasma viscosity is associated with higher rates of adverse clinical events in unstable angina pectoris and stroke (Kesmarky et al., 2008).

RBC Deformability

Red blood cell deformability is another important determinant of blood viscosity. RBC deformability depends on several factors, including internal (cytosolic) viscosity (mainly determined by the mean cell hemoglobin concentration), membrane viscoelasticity (which is dependent on cytoskeleton proteins and lipid bilayer properties), and the surface-area-to-volume ratio (also called cell sphericity) (Clark et al., 1983; Renoux et al., 2019). At low shear rates, rigid RBCs are less likely to aggregate than deformable RBCs. Therefore, a loss of RBC deformability at very low shear rates (less than 1 s^–1) results in a decrease in blood viscosity (Chien et al., 1970). In contrast, at shear rates above 1 s^–1, a decrease in RBC deformability causes blood viscosity to increase (Chien et al., 1970).

Initial experiments done to analyze RBC deformability in blood flow were conducted in the 1970s and 1980s using microtubes (Goldsmith et al., 1972) and rheoscopes (Fischer et al., 1978). These studies demonstrated that, as shear stress increases, normal RBCs align with the direction of flow by deforming into an elliptical shape via a “tank tread-like” motion of the cell membrane around the cytoplasm (Schmid-Schonbein et al., 1969; Goldsmith et al., 1972; Fischer et al., 1978). Rigid RBCs, on the other hand, cannot properly deform into an ellipse and remain perpendicular to blood flow, consequently increasing vascular resistance. In these fundamental experiments, RBCs were suspended in solutions, such as dextrose, with higher viscosities than the internal viscosity of a RBC (Goldsmith et al., 1972; Fischer et al., 1978). However, when a RBC is flowing in plasma in vivo, the plasma viscosity is lower than the viscosity of the erythrocyte’s cytosol. This is important because recent experiments reveal that the “tank-treading” behavior of erythrocytes does not occur when RBCs are suspended in solutions with lower viscosities that are more similar to plasma viscosity in vivo (Dupire et al., 2012). Instead, observations by Lanotte et al. (2016) showed that RBCs display a wide variety of cell shapes for any given flow condition. For example, erythrocytes in dilute suspensions behave like rigid oblate ellipsoids at low shear rates (<1 s^–1). Then, as shear rates increase, the erythrocytes successively tumble, roll, and deform into stomatocytes, and eventually adopt highly deformed poly-lobed. The findings of Lanotte et al. (2016) suggest that the pathological alterations of multiple parameters, including plasma composition, erythrocyte cytosol viscosity, and/or membrane mechanical properties could contribute to pathological blood rheology and flow. Further research should be conducted to determine how decreased RBC deformability could affect RBC shape transitions.

Red blood cell deformability is also a key determinant of blood flow in the microcirculation. RBCs are bi-concave disks with an average diameter of around 7–8 μm. Capillaries can have a diameter of less than 5 μm. Therefore, RBC must be highly deformable to pass through the narrowest vessels of the microcirculation. A 15% decrease in RBC deformability has been shown to cause a 75% increase in whole flow resistance in isolated perfused rat hind limbs (Baskurt et al., 2004). Moreover, when dog lungs were perfused with rigid RBC, whole pulmonary arterial pressure increased, and the main increase was localized in the microcirculation (Hakim, 1988; Raj et al., 1991). Indeed, any decrease of RBC deformability may affect flow resistance, tissue perfusion, and oxygenation (Parthasarathi and Lipowsky, 1999).

Sickle Cell Anemia as a Hemorheological Disease

Sickle cell anemia is caused by a single nucleotide mutation (adenine → thymine) in exon I of the β-globin gene. This point mutation (rs334 T) leads to the production of sickle hemoglobin (HbS), due to the substitution of valine for glutamic acid at the sixth position of the β-globin chain. The hydrophobic residue of valine associates with other hydrophobic residues, which causes HbS molecules to aggregate, forming fibrous precipitates when hemoglobin is deoxygenated. This phenomenon is called “HbS polymerization,” and is responsible for the characteristic shape change, termed “sickling,” of RBCs. Sickle RBC are rigid, and therefore do not easily flow through the microcirculation, causing frequent vaso-occlusive episodes in affected patients. In addition, when RBCs lose their deformability, they become more fragile and prone to hemolysis, which is the root cause of chronic hemolytic anemia in SCA (Connes et al., 2014).

Although the loss of RBC deformability is a fundamental characteristic of SCA, patients exhibit varying degrees of RBC rigidity, which can differentially affect SCA disease severity and complications (Renoux et al., 2016). Newborns usually have almost normal RBC deformability because they still have a high percentage of fetal hemoglobin (HbF) (Renoux et al., 2016). However, when patients become older HbF is replaced by HbS, and, as a result, RBC deformability decreases (Renoux et al., 2016). Hydroxyurea (HU) therapy can affect RBC deformability because HU stimulates HbF synthesis, thereby improving RBC deformability, although the deformability values still remain lower than in the healthy population (Lemonne et al., 2015). In individuals who are not under HU therapy, the presence or absence of alpha-thalassemia can also modulate RBC deformability (Renoux et al., 2017), as the inheritance of alpha-thalassemia results in decreased production of HbS (Rees et al., 2010) and thus lower HbS polymerization.

Rigid, sickle deformed RBC can cause pre-capillary obstruction and contribute to vaso-occlusion (Rees et al., 2010). Furthermore, RBC deformability further decreases during acute vaso-occlusive events. However, patients (adults and children over 5 years of age) who have the highest RBC deformability at steady-state, and who are not under HU therapy, actually have a higher risk of developing frequent painful vaso-occlusive crises, as well as osteonecrosis (Lamarre et al., 2012; Lemonne et al., 2013; Renoux et al., 2017). This paradox seems to be due to the fact that in SCA, RBC with the highest deformability are also the most adherent, which would trigger vaso-occlusion (Ballas et al., 1988). On the other hand, individuals who have improved RBC deformability as a result of HU therapy do not have an elevated risk of vaso-occlusive crisis because HU inhibits RBC, platelets and WBC adhesion to the vascular wall via several mechanisms (Bartolucci et al., 2010; Laurance et al., 2011; Chaar et al., 2014; Pallis et al., 2014; Verger et al., 2014). RBC aggregation is usually lower in SCA patients because of low hematocrit and the inability of irreversible sickle cell to form aggregates. However, the formed RBCs aggregates are reported to be much more robust than in healthy population which could further alter blood flow and tissue perfusion at the pre and post capillary levels (Tripette et al., 2009; Connes et al., 2014).

Sickle cell anemia patients with the highest degree of chronic hemolysis (i.e., those with the most severe anemia) are usually characterized by a severe and chronic reduction of RBC deformability as well as the presence of robust RBC aggregates, and seem to be more prone to develop complications such as leg ulcers, priapism and glomerulopathy (Bartolucci et al., 2012; Connes et al., 2013a; Lamarre et al., 2014). In contrast, patients with the highest RBC deformability tend to have less severe anemia and increased blood viscosity, which could increase the risk of developing frequent vaso-occlusive like complications (Nebor et al., 2011; Lamarre et al., 2012; Charlot et al., 2016). Surprisingly, a recent study did not show a further rise of blood viscosity during acute painful vaso-occlusive crisis (Lapoumeroulie et al., 2019). Instead, the authors found a decrease of RBC deformability, as a result of massive RBC sickling (Ballas and Smith, 1992), and a rise in RBC aggregation and RBC aggregates robustness, which would probably impair blood flow into the microcirculation (Lapoumeroulie et al., 2019).

The Effect of Acute Cycling Exercise on Blood Viscosity and Its Determinants in Healthy Individuals

Most of the studies conducted in the eighties and nineties to investigate the acute effects of exercise on blood rheology focused on cycling efforts performed by moderately trained subjects (Brun et al., 1998). These studies reported that acute cycling exercise caused blood viscosity measured at high shear rate (i.e., 100–200 s^–1) to increase by more than 15% above pre-exercise values (Brun et al., 1998; Connes et al., 2004a, 2013b; Figure 1). This increase in blood viscosity has been attributed to changes in plasma viscosity, hematocrit, and RBC rheological properties (Brun et al., 1998).

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FIGURE 1

Effects of different kind of exercise on blood viscosity measured at several shear rates in the same trained subject (maximal oxygen consumption, VO₂max = 64 ml/kg/min). The maximal treadmill and cycling tests consisted in a progressive and maximal exercise conducted until VO₂max and performed in laboratory conditions (temperature: 24°C). The 10 km running was performed outdoor (20°C) and at the highest intensity the subject could run. Indeed, the subject ran at 77% of his maximal aerobic velocity determined on the treadmill and reached a heart rate of 92% of his maximal heart rate. The subject did not drink water during each test. For the three tests, blood was sampled immediately at the end of the exercise and blood viscosity was measured with the same cone-plate viscometer within 1 h after sampling.

Multiple studies have shown that an acute bout of maximal or submaximal cycling exercise may cause plasma viscosity to increase by 10–12%, compared to resting values (Ernst, 1985; Ernst et al., 1991b; Brun et al., 1994b, 1998; Bouix et al., 1998; Connes et al., 2004b). This increase has been attributed to a rise in plasma protein content, such as fibrinogen, α1-globulins, α2-globulins, β-globulins, and γ-globulins during exercise (Nosadova, 1977; Convertino et al., 1981; Vandewalle et al., 1988; Wood et al., 1991).

Several studies have also shown that an acute cycling exercise can result in a 10–12% (i.e., 3–4 units) rise in hematocrit, when compared to pre-exercise values (Brun et al., 1994b; Bouix et al., 1998; Connes et al., 2004a). The effect of elevated hematocrit on blood viscosity at high shear rates has been well described (Cokelet and Meiselman, 2007). A 1-unit increase in hematocrit can cause a 4% increase in blood viscosity. This rise in hematocrit has been attributed to several mechanisms such as fluid shift between intra- and extra-vascular spaces (Sjogaard et al., 1985; Ploutz-Snyder et al., 1995), dehydration (Nosadova, 1977; Stephenson and Kolka, 1988) the release of sequestered RBCs from the spleen (Isbister, 1997), and water trapping in muscle (Ploutz-Snyder et al., 1995).

Acute cycling exercise can also modulate RBC deformability. Several authors have reported a decrease in RBC deformability during and immediately after exercise (Reinhart et al., 1983; Galea and Davidson, 1985; Gueguen-Duchesne et al., 1987; Brun et al., 1993, 1994a; Oostenbrug et al., 1997; Yalcin et al., 2000, 2003). This decrease could be a consequence of lactic acid and reactive oxygen species production that occurs during exercise (Connes et al., 2013b). The accumulation of lactate ions and the decrease in pH would promote RBC dehydration through the activation of several RBC cationic channels, leading to RBC shrinkage and decreased RBC deformability (Van Beaumont et al., 1981; Lipovac et al., 1985; Smith et al., 1997; Connes et al., 2004c). Moreover, several groups have suggested that oxidative stress could also play a role in the decreased RBC deformability through the oxidation of membrane lipid and protein components (Senturk et al., 2005a, b; Connes et al., 2013b). However, the magnitude of the change in RBC deformability that occurs during exercise seems to depend on the training status and physical fitness of the subjects. For instance, Senturk et al. (2005a) reported that a short, progressive, maximal cycling exercise test promoted oxidative stress in both sedentary and well-trained subjects, but they only observed a decrease in RBC deformability and an increase of RBC fragility in sedentary individuals.

Very few studies have observed RBC aggregation during cycling exercise, and the results of these studies are very heterogeneous (Connes et al., 2013b). Yalcin et al. (2003) found that RBC aggregation decreased after a Wingate exercise test. In contrast, Ernst et al. (1991b) observed a transient increase in RBC aggregation above pre-exercise values after 1 h of pedaling at a heart rate of 150 beats min^–1. RBC aggregation then returned to baseline during the 2 h following the cycling test. Varlet-Marie et al. (2003) and Connes et al. (2007) found no change in RBC aggregation in response to submaximal/maximal cycling exercise. The discrepancies observed are not very well understood and might be related to the (1) kind of exercise performed (short versus prolonged exercise), (2) time of measurement during exercise (i.e., immediately at the end of exercise or few minutes after), (3) time delay for measurement after sampling, and (4) procedure used for RBC aggregation measurement (adequate oxygenation and adjusted hematocrit before measurement or not).

On the whole, these changes lead to a rise in blood viscosity during acute cycling exercise. Large elevations in blood viscosity have generally been considered to be deleterious for the cardiovascular system (Brun et al., 1998; Connes et al., 2013b). However, Connes et al. (2012) reported a positive correlation between the magnitude of change in blood viscosity and the magnitude of change in NO end-stable products, and a negative correlation between the magnitude of change in blood viscosity and the magnitude of change in vascular resistance. These findings suggest that increasing blood viscosity during exercise could be a way to stimulate endothelium-dependent NO production through shear stress-related mechanisms. In healthy individuals, this would result in compensatory vasodilation, thereby preventing any large increases in vascular resistance.

Acute Running Exercise, Blood Viscosity and Its Determinants in Healthy Individuals

Surprisingly, in contrast to cycling exercises, running exercises, such as a marathon or a 10-km race, do not cause a rise in blood viscosity (Neuhaus et al., 1992; Tripette et al., 2011; Figure 1). The main reason is that hematocrit and plasma viscosity usually remain very stable during these kinds of efforts, despite dehydration (Galea and Davidson, 1985; Neuhaus et al., 1992; Neuhaus and Gaehtgens, 1994). It has been hypothesized that the lack of change in hematocrit could be explained by repeated RBC foot strike hemolysis during running (Neuhaus et al., 1992; Neuhaus and Gaehtgens, 1994; Tripette et al., 2011; Connes et al., 2013b). However, a recent study in which highly trained subjects performed a progressive and maximal treadmill test did not observe any signs of RBC damage or eryptosis (Nader et al., 2018). However, the picture could be slightly different during ultra-running events. For instance, Robach et al. (2014) reported increased hemolysis and large plasma volume expansion immediately after a 166-km long mountain ultra-endurance marathon with 9500 m of altitude gain/loss. The impact of these changes on blood rheology has not been investigated and further studies are needed to address this question.

Surprisingly, Nader et al. (2018) found a slight but significant increase in RBC deformability in response to a short and maximal exercise test. Indeed, although hematocrit increased, blood viscosity remained unchanged (and tended to decrease), as has been observed in previous studies (Neuhaus and Gaehtgens, 1994; Tripette et al., 2011). The findings suggest that the slight increase in RBC deformability could have compensated for the rise in hematocrit observed during short running events, resulting in a lack of change in blood viscosity. The impact of ultra-run events on RBC deformability is unknown. Several other groups found a slight increase in RBC deformability during running (Suhr et al., 2012) and cycling (Connes et al., 2009) exercises. Although the exact reasons for these findings are unknown, it has been suggested that increased NO production during exercise could increase RBC deformability. One of the first works to suggest that NO could affect RBC deformability was the study of Starzyk et al. (1997), which demonstrated that intravenous infusion of L-NAME (an eNOS inhibitor) in rats caused a reduction of RBC deformability. In addition, Bor-Kucukatay et al. (2003) reported that several eNOS inhibitors also decreased RBC deformability, suggesting that basal release of NO actively maintains RBC deformability. While extracellular sources of NO may impact the deformability of RBC, several works suggest that endogenous RBC NO synthesis may also modulate RBC deformability (Kleinbongard et al., 2006). Suhr et al. (2012) demonstrated that an acute running exercise induced shear stress activation of RBC NOS (increased RBC NOS phosphorylation at Ser1177) via the PI3-kinase/Akt kinase pathway, leading to increased NO production by the RBC, which was critical to maintaining RBC deformability during exercise. Grau et al. (2013) further extended these findings by showing that RBC NOS activation by pharmacological treatment (insulin) increased RBC NO content and improved RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely the α- and β-spectrins. In contrast, the use of RBC NOS inhibitors [wortmannin or L-N5-(1-Iminoethyl)-ornithin] resulted in a decrease of RBC NOS Ser1177 phosphorylation, NO content, cytoskeleton protein S-nitrosylation, and RBC deformability.

Impact of Physiological Changes During Acute Exercise on Blood Viscosity

As discussed above, acute cycling exercise would lead to a rise in blood viscosity while running exercise would be characterized by a lack of change in blood viscosity compared to the pre-exercise values. Although methodological aspects could partly explain some of these differences, such as the use of different viscometers (cone-plate viscometer, couette viscometer, capillary viscometer, etc.) or the use of different shear rates (low, moderate, and high shear rate viscosity are affected by different RBC rheological parameters), the picture is probably a little bit more complex. Several physiological changes occur during exercise, which may affect blood viscosity.

To avoid large dehydration during exercise, sportsmen usually drink water or carbohydrate-rich drinks. However, most of the studies performed in the field of blood rheology have been done in laboratory conditions where water is not allowed during the various exercise tests and water loss cannot be compensated by water intake. Tripette et al. (2010) and Diaw et al. (2014) previously tested the impact of add-libitum hydration and water deprivation on blood viscosity during a prolonged submaximal exercise and a soccer game, respectively. Healthy individuals and subjects carrying sickle cell trait were included. While hydration during exercise was able to decrease blood viscosity below the pre-exercise levels in sickle cell trait carriers, blood viscosity increased similarly in healthy individuals in both the “hydration” and “water deprivation” conditions. The rate of dehydration was around 1.5–2% in these studies, which is not very severe. There is a growing interest from the runners community to participate to very prolonged race (>100 kms), sometimes performed at high altitude. The impact of the environment and higher dehydration rate on blood viscosity are unknown and further studies are needed.

Exercise is accompanied by a rise cardiac output and blood flow leading to an increase of shear rate values in the vascular system. For instance, shear rate in the femoral artery has been reported to increase from 60 s^–1 at rest to 200–250 s^–1 during exercise (Gonzales et al., 2009). Blood is a shear-thinning fluid, meaning that its viscosity decreases when shear rate increases. Connes et al. (2013b) shown that 15 min of cycling exercise performed at a submaximal intensity increased blood viscosity measured at 90 s^–1. However, when the viscosity of the blood sampled at the end of exercise was measured at 225 s^–1 (which reflects the shear rate reached during exercise), the mean value was almost identical to the viscosity of the blood sampled before exercise and measured at 90 s^–1. Indeed, the effects of hemoconcentration, increased plasma viscosity, decreased RBC deformability and increased RBC aggregation on blood viscosity during exercise could be counterbalanced by the effects of increasing shear rate (Connes et al., 2013b). However, the slight remaining changes in blood viscosity observed during exercise were still correlated with the magnitude of changes in NO-end products concentration, suggesting that blood viscosity plays a role in promoting NO production during exercise (Connes et al., 2013b).

Core temperature rises during exercise and it is well known that blood viscosity also depends on temperature (Baskurt et al., 2009). A recent elegant study shown that when the changes in temperature occurring during exercise are took into account in the measurements of blood viscosity, there is no difference between the pre- and post-exercise values (Buono et al., 2016). The slight physiological hyperthermia was shown to increase RBC deformability, which compensated the rise in hematocrit and resulted in a lack of change in blood viscosity. The authors estimated that the combined effects of increasing shear rate and hyperthermia during exercise could decrease blood viscosity by 31% below the pre-exercise levels, in spite of the exercise induced hemoconcentration (Buono et al., 2016). These findings clearly show that studies in the field of exercise hemorheology should consider the effects of various physiological factors for better interpretation about the role of blood viscosity in the cardiovascular adaptations and physical fitness.

Long Term Effects of Exercise on Blood Rheology in Healthy Individuals

Chronic exercise (endurance or resistance exercise) usually decreases blood viscosity (Brun et al., 1998; Romain et al., 2011; Kilic-Toprak et al., 2012). One reason for this change is that plasma volume increases few hours or several days after a single bout of exercise (Fellmann, 1992; Brun et al., 1998), resulting in an “autohemodilution” (Ernst, 1987; Ernst et al., 1991a). Repeated exercise bouts on consecutive days leads to a chronic “autohemodilution” resulting in a low baseline hematocrit – low baseline viscosity pattern (Ernst, 1987; Brun et al., 1998; Kilic-Toprak et al., 2012). The magnitude of plasma volume expansion ranges from 9 to 25%, corresponding to an additional 300 to 700 ml of plasma. It has been shown that the larger the reduction in plasma volume during exercise, the greater the subsequent plasma volume expansion (Fellmann, 1992). The amount of water ingested during and after exercise, as well several fluid-regulating hormones (aldosterone, vasopressin and the atrial natriuretic factor) and the level of plasma proteins, influence the degree of the plasma volume expansion after exercise. Plasma volume expansion is responsible for the decrease in plasma viscosity that contributes to the decrease in blood viscosity (Ernst, 1987).

Exercise training also induces RBC rheological adaptations (Brun et al., 2010; Kilic-Toprak et al., 2012). Ernst (1987) reported increased RBC deformability in athletes compared to sedentary subjects, a finding later confirmed by Smith et al. (1999). A 3-month longitudinal study of initially untrained healthy volunteers who performed regular training also showed a decrease of blood viscosity and a rise of RBC deformability (Ernst, 1987). Few studies have been conducted to determine why RBC deformability improves in healthy individuals after chronic exercise. However, Smith et al. (1999) and Tomschi et al. (2018) found a higher proportion of young deformable RBCs in athletes than in untrained subjects. The hemorheological benefits induced by regular exercise are suspected to contribute to the improvements in cardiovascular health that are induced by training programs in patients with cardiovascular disorders (Sandor et al., 2014).