Plasmodium berghei: the influence of blood volume changes on the malaria-induced anemia in Balb/C mice

Malaria-induced anemia exceeds that attributable to direct parasite destruction of erythrocytes. Since spleen and liver weights increase significantly, hemodilution may account for part of this excessive anemia. To determine the role of hemodilution in the etiology of anemia, vascular volumes were measured with Evans blue and isotope dilution techniques. The Evans blue dilution technique showed that blood volume increased 20%, in infected Balb/C mice. However, when blood volume was measured with Evans blue bound to protein prior to injection or with iodinated albumin and chromium-labelled red blood cells no significant change was detected. Evidently the permeability of the vasculature and/or erythrocytes of infected mice was increased so that injected Evans blue occupied a space larger than the vascular volume before becoming bound to plasma proteins. We conclude that hemodilution is not involved in the excessive anemia of Plasmodium berghei-infected Balb/C mice.     

Splenic respiratory compensation and blood volume in the newt

In the newt Triturus cristatus carnifex , reversible increase in size of the spleen is linked to the respiratory state of the animal: when the newt is exposed to the air, and thus well oxygenated, the spleen hoards erythrocytes; when immersed in still water, in an hypoxic state, the spleen releases erythrocytes into the bloodstream. In chlorobutanol-anaesthetized specimens exposed to the air, the maximum size reached by the spleen diminishes with a rise in temperature up to the disappearance of all congestion at 33°C. The blood volume of newts kept in humid air at 6°C and 18°C after anaesthesia varies from about 6–9 ml per 100 g of body weight, while the red blood cell count and haematocrit value remain stable. In anaesthetized specimens kept in still water at the same temperatures the blood volume is stable, at about 7 ml per 100 g, but the red cell count and haematocrit are notably higher. At 33°C, a critical temperature for the newts, the specimens in still water succumb while those in air present the same blood volume as at 18°C, but have a higher erythrocyte count and haematocrit value.     

Haematopoietic stem cell content of murine bone marrow, spleen, and blood. Limiting dilution analysis of diffusion chamber cultures

Suspensions of mouse bone marrow cells, spleen cells, and blood leucocytes were cultured in diffusion chambers in dilution series in order to establish the minimum concentrations of haematopoietic stem cells (HSC). The observed frequencies of empty chambers after seven days of culture conformed to the expected frequencies of a null response in a Poisson distribution. The proportions of empty chambers could therefore be used to estimate the concentrations of HSC in the cell suspensions. The following numbers of HSC per 10⁵ cells were found (with 95% confidence limits): Bone marrow: 50 (44–56). Spleen: 3.5 (2.8–4.3). Blood leucocytes: 1.4 (1.2–1.8). The mean (± standard error) HSC-content per femur, spleen, and milliliter blood when pooling cells from three to six donor mice was 8240 ± 600, 7660 ± 490, and 56 ± 6.5 respectively. For comparison, the HSC concentrations were also determined with the spleen colony technique; the ratio between the HSC-concentrations of bone marrow, spleen, and blood determined with the diffusion chamber technique was similar to that determined with the spleen colony technique.     

Deglycerolization of red blood cells: A new dilution-filtration system

In this work, we present a new version of the dilution-filtration system for rapidly deglycerolizing a large volume of cryopreserved blood. In our earlier system, one of the major problems was the damage induced to the red blood cells (RBCs) due to high osmolality change at the dilution point. Therefore, we devised a new system to solve this problem. First, we theoretically simulated the osmolality variation in the new system and the variation of the maximum and minimum volumes of the RBCs at the dilution point to examine the effects of operating parameters/conditions. Next, we experimentally validated the effects of these operating parameters by deglycerolizing porcine blood. The results show that when the initial NaCl concentration in the hypertonic solution is 18%, the volume of the hypertonic solution is 200 mL, and the flow rate of the filtrate is 50 mL/min, the system can effectively remove glycerin from 200 mL of porcine blood in 30 min, with ∼87% RBC survival rate and ∼73% RBC recovery rate. Our results indicated that in the new system the concentration and the volume of the hypertonic solution used to dilute the blood are the important parameters that need to be adjusted to reduce osmotic damage to the RBCs. In addition, a fast filtrate flow rate is highly recommended. This work can significantly contribute to the development of a more efficient and effective system for deglycerolizing large volumes of cryopreserved blood in clinic.     

Tissue blood content in anaesthetised sheep and horses

The blood content of tissue in eight anaesthetized sheep and three anaesthetized horses was measured using chromium labelled autologous red blood cells. 2. Approximately half the total blood volume was found to be distributed within the body tissues and, with the exception of the spleen and fat, the distribution was similar in these two species.     

Reciprocal splanchnic-thoracic blood volume changes during the Valsalva maneuver

The Valsalva maneuver is frequently used to test autonomic function. Previous work demonstrated that the blood pressure decrease during the Valsalva maneuver relates to thoracic hypovolemia, which may preclude pressure recovery during phase II, even with normal resting peripheral vasoconstriction. We hypothesized that increased regional blood volume, specifically splanchnic hypervolemia, accounts for the degree of thoracic hypovolemia during the Valsalva maneuver. We studied 17 healthy volunteers aged 15-22 yr. All had normal blood volumes by dye dilution. Subjects also had normal vascular resistance while supine as well as normal vasoconstrictor responses during 35 degrees upright tilt. We assessed changes in estimated splanchnic, pelvic-thigh, and lower leg blood volume, along with thoracic blood volume shifts, by impedance plethysmography before and during the Valsalva maneuver performed in the supine position. Early increases in splanchnic blood volume dominated the regional vascular changes during the Valsalva maneuver. The increase in splanchnic blood volume correlated well (r2 = 0.65, P < 0.00001) with the decrease in thoracic blood volume, there was less correlation of the increase in pelvic blood volume (r2 = 0.21, P < 0.03), and there was no correlation of the increase in leg blood volume (r2 = 0.001, P = 0.9). There was no relation of thoracic hypovolemia with blood volume or peripheral resistance in supine or upright positions. Thoracic hypovolemia during the Valsalva maneuver is closely related to splanchnic hyperemia and weakly related to regional changes in blood volume elsewhere. Changes in baseline splanchnic vascular properties may account for variability in thoracic blood volume changes during the Valsalva maneuver.     

Differential T cell responses to Plasmodium chabaudi chabaudi in peripheral blood and spleens of C57BL/6 mice during infection

The definition of the immune status of a person is often taken as the responses obtained from lymphocytes isolated from peripheral blood. We therefore analyzed in a mouse model of Plasmodium chabaudi chabaudi the response of T lymphocytes taken from peripheral blood and compared it with the spleen during and after a primary erythrocytic infection. Using limiting dilution conditions, no malaria-specific T cell responses could be measured in the peripheral blood for up to 21 days after infection with P. chabaudi, whereas T cells responding to malaria Ag were readily detected in the spleen. This was true for T cells providing help and for those producing IFN-gamma. After clearance of the parasitemias to subpatent levels (75 days), qualitatively similar T cell responses were found in both compartments of the immune system, i.e., the Th cell response predominated over the inflammatory response. These data suggest that during an active infection with Plasmodium, T cell responses in peripheral blood are not necessarily indicators of the immune status.     

Particle-induced myeloperoxidase release in serially diluted whole blood quantifies the number and the phagocytic activity of blood neutrophils and opsonization capacity of plasma.

Luminol-amplified chemiluminescence (CL) from phagocytes has previously been shown to be almost completely dependent on the release of myeloperoxidase (MPO) from azurophilic granules. We measured the luminol-amplified chemiluminescence response (WBCL) by using serially diluted whole blood. In these experiments, non-opsonized and serum-opsonized zymosan (NWBCL and OWBCL, respectively) were used concurrently as phagocytosable particles. We found two whole-blood dilution ranges with clinical significance: first, <0.04% of whole blood in the reaction volume, where MPO released by the zymosan-activated leukocyte population came almost totally from neutrophils and the OWBCL response could be exploited as a measure of a neutrophil count in a given blood specimen, despite the pathophysiological state of the host. In contrast, the NWBCL response was two-fold in blood samples from bacterial infection patients compared to those of controls and patients with viral infection, suggesting the use of NWBCL for the differential diagnosis of bacterial infections from viral infections; second, 0.16-1.2% of whole blood in the reaction volume, where the opsonization capacity of plasma (OC(50)) can be determined. We also found that at whole blood content >0.04%, erythrocytes quickly start to absorb chemiluminescence light, and that at whole blood content >1.2%, plasma proteins, most probably albumin and fibrinogen, start to inhibit MPO release.     

Measurement of blood volume in the Indian mud eel Amphipnous cuchia (Ham.) (amphipnoidae; pisces).

The blood volume of 10 male and 8 female Amphipnous cuchia (Ham.) were measured through dilution technique method using Evans blue and sodium citrate as anticoagulant. This method was established against heparin. The mean total blood volume and the blood volume per 100 g of body weight for all the weight groups in male were 9.73 +/- 2.45 and 3.10 +/- 0.66, and in female were 10.34 +/- 2.78 and 3.13 +/- 0.66 respectively. The total blood volume of male and female fishes increase as an exponential function of its total body weight.     

Effect of the chest wall and blood volume on pulmonary distensibility.

To determine the reason for increased pulmonary distensibility in excised lungs, we performed deflation pressure-volume (PV) studies in 24 dogs. Exponential analysis of PV data gave K, an index of distensibility. Lung volume was measured by dilution of neon. Compared with measurements obtained in the supine position, with the chest closed, and with esophageal pressure (Pes) to obtain transpulmonary pressure, K was not changed significantly with the chest strapped, with pleural pressure to obtain transpulmonary pressure, or with the chest open. From displacement of PV curves obtained in the supine position and with the chest closed or open, we estimated that Pes was 0.18 kPa greater than average lung surface pressure. An increase in K in the prone and head-up positions was attributed to a traction artifact decreasing Pes. Exsanguination increased K and produced a relative increase in gas volume. These results show that overall pulmonary distensibility is unaffected by an intact chest wall. An increase in K and gas volume after exsanguination probably reflects a decreased pulmonary blood volume, with collapse of capillaries increasing the alveolar volume-to-surface ratio.     

Red cell and total blood volumes during sexual excitement and copulation in the boar.

Measurements of hematocrit, total plasma protein and red cell volume were made during sexual excitement and copulation in boars. Red cell volume (RCV) was determined by isotope dilution technique using endogenous 51Cr-tagged red cells. Basing on these data changes in the total blood volume (TBV) and plasma volume (PV) were calculated by 2 indirect methods. RCV increased by 12% to 16% in the intial phase of ejaculation and remained increased during ejaculation and 40 minutes after copulation. TBV and PV decreased during copulation, greatest drop being found in the final phase of ejaculation. After ejaculation the TBV was increasing, first to the resting value before copulation (about 20 minutes after copulation), thereafter it became markedly higher than during the resting period. Depending on the method used for calculation significant differences were found in the quantity of TBV and PV drop during ejaculation.     

Ethanol consumption and blood pressure

Chronic ethanol consumption consistently resulted in mild hypertension in the male Wistar rats used in the present study. The ethanol-treated animals also have reduced 24-hour urinary output and significant sodium retention when compared to the controls. Total plasma volume was estimated using the technique of indicator dilution, and an increase of 20% was observed in the ethanol-treated animals. Vascular smooth muscle resposiveness to noradrenaline in vitro was not different between the two groups of animals. Therefore the blood pressure elevation in the ethanol-treated animals seems to be associated with sodium retention and plasma volume expansion, and probably is unrelated to altered vascular responsiveness.     

Changes in regional blood volume and blood flow during static handgrip

Increased blood pressure (BP) and heart rate during exercise characterizes the exercise pressor reflex. When evoked by static handgrip, mechanoreceptors and metaboreceptors produce regional changes in blood volume and blood flow, which are incompletely characterized in humans. We studied 16 healthy subjects aged 20-27 yr using segmental impedance plethysmography validated against dye dilution and venous occlusion plethysmography to noninvasively measure changes in regional blood volumes and blood flows. Static handgrip while in supine position was performed for 2 min without postexercise ischemia. Measurements of heart rate and BP variability and coherence analyses were used to examine baroreflex-mediated autonomic effects. During handgrip exercise, systolic BP increased from 120 +/- 10 to 148 +/- 14 mmHg, whereas heart rate increased from 60 +/- 8 to 82 +/- 12 beats/min. Heart rate variability decreased, whereas BP variability increased, and transfer function amplitude was reduced from 18 +/- 2 to 8 +/- 2 ms/mmHg at low frequencies of approximately 0.1 Hz. This was associated with marked reduction of coherence between BP and heart rate (from 0.76 +/- 0.10 to 0.26 +/- 0.05) indicative of uncoupling of heart rate regulation by the baroreflex. Cardiac output increased by approximately 18% with a 4.5% increase in central blood volume and an 8.5% increase in total peripheral resistance, suggesting increased cardiac preload and contractility. Splanchnic blood volume decreased reciprocally with smaller decreases in pelvic and leg volumes, increased splanchnic, pelvic and calf peripheral resistance, and evidence for splanchnic venoconstriction. We conclude that the exercise pressor reflex is associated with reduced baroreflex cardiovagal regulation and driven by increased cardiac output related to enhanced preload, cardiac contractility, and splanchnic blood mobilization.     

Haematology of swordtail, Xiphophorus helleri. I: blood parameters and light microscopy of blood cells

The main blood parameters of the swordtail, Xiphophorus helleri , were studied. Morphology, granulation staining and cytochemistry of leucocytes in peripheral blood, kidney, spleen and gills were investigated by light microscopy. Blood parameters are similar to other fish species: Red blood cell count (4.5 × 10⁶μl), white blood cell count (15.2 × 10³μl), haema-tocrit (33.8%) haemoglobin (7.8 mg ml⁻¹), MCV (mean corpuscle volume, 75.1 μm³). MCH (mean content of haemoglobin, 17.3 pg), MCHC (mean percentage haemoglobin/erythrocyte, 23.1%/100 ml erythrocytes). Leucocytes can be classified into lymphocytes, thrombocytes, neutrophilic and eosinophilic gra-nulocytes, monocytes macrophages and melanomacrophages.
Morphological and cytochemical features of the cells are described and compared with results from other fish species.     

A realistic evaluation of the action of ozone on whole human blood

We have clarified the role of the ozone concentration in relation to the resistance of human erythrocytes in whole human blood or in blood diluted either in saline or in distilled water.

Spectrophotometric data related to haemoglobin were evaluated by exposing samples of fresh human blood directly to ozone doses (ratio 1:1 volume), within the therapeutic range (0.21–1.68 mM) and to one toxic dose (3.36 mM). Furthermore, the same determinations have been carried out after previous dilution of the same blood with either pure water or physiological saline (1 ml blood + 29 ml diluent) followed by ozonation with the above reported ozone doses. Addition of either saline or water implies a dilution of plasma antioxidants and also total haemolysis after water dilution. Particularly the latter case represents a most unphysiological situation because the osmotic shock causes the solubilization of the erythrocytic content. While it is possible to demonstrate that after haemolysis there is an ozone-concentration dependent transformation of some oxyhaemoglobin to methaemoglobin, no such process occurs after ozonation of whole blood.

The results of this study fully confirm our previous data that judicious ozone doses neither damage erythrocytes, nor induce oxidation of intracellular haemoglobin. We hope that our conclusions will definitively clarify the absence of toxicity of ozonetherapy.     

Cardiovascular and splenic responses to exercise in humans.

To investigate splenic erythrocyte volume after exercise and the effect on hematocrit- and hemoglobin-based plasma volume equations, nine men cycled at an intensity of 60% maximal O(2) uptake for 5-, 10-, or 15-min duration, followed by an incremental ride to exhaustion. The reduction in spleen volume, calculated using (99m)Tc-labeled erythrocytes, was not significantly different among the three submaximal rides (5 min = 28%, 10 min = 30%, 15 min = 36%; P = 0.26). The incremental ride to exhaustion resulted in a 56% reduction in spleen volume, which recovered to baseline levels within 20 min. Plasma catecholamines were inversely related to spleen volume after exercise (r = 0.70-0.84; P < 0.0001). There were no differences in red cell or total blood volume pre- to postexercise; however, a significant reduction in plasma volume was observed (18.9%; P < 0.01). There was no difference between the iodinated albumin and the hematocrit and hemoglobin methods of assessing plasma volume changes. These results suggest that the spleen regulates its volume in response to an intensity-dependent signal, and plasma catecholamines appear partially responsible. Splenic release of erythrocytes has no effect on indirect measures of plasma volume.     

The cellular composition of the blood and haematopoietic organs of turbot Scophthalmus maximus L.

The cellular composition of the blood, anterior kidney, spleen and thymus of turbot Scophrhalmus maximus L., aged 1 + was determined. Ninety-four per cent of blood cells belonged to the erythrocyte lineage of which 82% were mature erythrocytes. The leucocytes, which represented 4.5% of the blood cells, were mainly lymphocytes (50%). The presence of crythroblasts in the anterior kidney and the spleen demonstrated an erythropoietic activity in both organs. However, this activity appeared to be prevalent in the spleen which also appeared to act as a storage zone for erythrocytes and as the centre point for thrombopoiesis. Although 96% of the anterior kidney cells were leucocytes, the number of white cells per gram of organ was higher in the spleen.     

Splenic hemodynamics and decreased endothelial nitric oxide synthase in the spleen of rats with liver cirrhosis

The enlarged spleen in liver cirrhosis is considered to play a role in the pathogenesis of portal hypertension, but the splenic hemodynamics and molecular mechanisms behind the phenomenon have not been elucidated. The present study aimed to examine the splenic hemodynamics associated with splenic microcirculation and congestion, and to determine the status of the endothelial nitric oxide synthase (eNOS) signaling pathway in the spleen of rats with liver cirrhosis. Liver cirrhosis was induced by bile duct ligation. In rats with bile duct ligation (BDL rats) and control rats, splenic blood flow was measured using a laser Doppler flowmeter, and splenic blood volume was measured using a near-infrared spectrophotometer. The expressions of eNOS and its upstream effectors, Akt, TNF-alpha and VEGF, in the spleen were also determined. Specific splenic blood flow was significantly decreased in BDL rats compared with control rats. Specific splenic blood volume was also decreased in BDL rats, while their total splenic blood volume, especially the deoxygenated volume, was significantly increased. The expressions of phosphorylated and total eNOS, and the eNOS phosphorylation ratio, were all significantly decreased in the spleen of BDL rats. The Akt phosphorylation ratio and TNF-alpha concentration were also decreased in the spleen of BDL rats although the expression of VEGF was increased. These findings suggest that the eNOS signaling pathway is suppressed in the spleen of cirrhotic rats, and may contribute to the measured decreases in specific blood flow and volume in the spleen of liver cirrhosis. Determination of the factors influencing the suppression of eNOS in the spleen may shed light on how liver cirrhosis results in hypodynamic intrasplenic circulation.     

Role of spleen in ANF-induced reduction in plasma volume.

Atrial natriuretic factor (ANF) causes an increase in hematocrit that cannot be accounted for by urinary losses. The mechanism behind this phenomenon was studied in intact and splenectomized rats. Rat ANF 99-126 was infused i.v. for 30 min into conscious rats at rates of 0 (saline control), 0.05, or 0.1 microgram/min. Plasma volume was then determined by dilution of the dye, Evan's Blue. In one group of rats, red cell volume was determined using 51Cr-labelled erythrocytes. ANF infusion was continued uninterrupted throughout the experiments. In the intact rats, ANF (0.10 microgram/min) caused hematocrit to increase from 38.9 +/- 0.5 to 41.2 +/- 0.4% (p < 0.005). Splenectomy so attenuated this response to ANF that it failed to reach significance. Similarly, ANF (0.10 microgram/min) caused plasma volume to fall from 5.1 +/- 0.1 to 4.5 +/- 0.1 mL/100 g body wt. (p < 0.005) in the intact rats, but did not affect plasma volume in the splenectomized rats. As a result, blood volume was significantly reduced by ANF in the intact rats, but remained unchanged in the splenectomized rats. Red cell volume did not change in response to infusion of ANF, nor did ANF affect the rate of clearance of Evan's Blue out of the plasma. It is concluded that the spleen is an important site of movement of protein-poor fluid out of the vasculature, and that this exchange is influenced by ANF.     

Significance of plasma skimming and plasma volume expansion.

The organs associated with plasma volume expansion, i.e., the red bone marrow, the enlarged spleen, and the uteroplacental complex, are arteriovenous shunts with an interposed sinusoidal stroma able to skim off plasma-rich blood. In the spleen, plasma separation is an integral part of the hemoconcentration. In the red bone marrow, plasma skimming might provide a washout mechanism for the many newly formed erythrocytes and platelets from the sinusoids to the peripheral blood circulation. In the uteroplacental complex, skimming of plasma-rich blood is beneficial in increasing blood flow in the myometrium, kidneys, and skeletal musculature. The hypervolemic status with anemia will simulate a negative iron balance, which speeds up the absorption of iron. Thus a conceptual unit seems to exist in which rheological factors influence such functions as transport of newly formed blood cells into the circulation (in the red bone marrow), hemoconcentration (in the spleen), and iron balance during pregnancy (in the uteroplacental complex).