Passage Of Erythrocytes Research Articles

O2 permeability of lipid bilayers is low, but increases with membrane cholesterol

Oxygen on its transport route from lung to tissue mitochondria has to cross several cell membranes. The permeability value of membranes for O2 (PO2), although of fundamental importance, is controversial. Previous studies by mostly indirect methods diverge between 0.6 and 125 cm/s. Here, we use a most direct approach by observing transmembrane O2 fluxes out of 100 nm liposomes at defined transmembrane O2 gradients in a stopped-flow system. Due to the small size of the liposomes intra- as well as extraliposomal diffusion processes do not affect the overall kinetics of the O2 release process. We find, for cholesterol-free liposomes, the unexpectedly low PO2 value of 0.03 cm/s at 35 °C. This PO2 would present a serious obstacle to O2 entering or leaving the erythrocyte. Cholesterol turns out to be a novel major modifier of PO2, able to increase PO2 by an order of magnitude. With a membrane cholesterol of 45 mol% as it occurs in erythrocytes, PO2 rises to 0.2 cm/s at 35 °C. This PO2 is just sufficient to ensure complete O2 loading during passage of erythrocytes through the lung’s capillary bed under the conditions of rest as well as maximal exercise.

Validation of red blood cell flux and velocity estimations based on optical coherence tomography intensity fluctuations

We present a validation of red blood cell flux and speed measurements based on the passage of erythrocytes through the OCT’s focal volume. We compare the performance of the so-called RBC-passage OCT technique to co-localized and simultaneously acquired two-photon excitation fluorescence microscopy (TPEF) measurements. Using concurrent multi-modal imaging, we show that fluctuations in the OCT signal display highly similar features to TPEF time traces. Furthermore, we demonstrate an overall difference in RBC flux and speed of 2.5 ± 3.27 RBC/s and 0.12 ± 0.67 mm/s (mean ± S.D.), compared to TPEF. The analysis also revealed that the OCT RBC flux estimation is most accurate between 20 RBC/s to 60 RBC/s, and is severely underestimated at fluxes beyond 80 RBC/s. Lastly, our analysis shows that the RBC speed estimations increase in accuracy as the speed decreases, reaching a difference of 0.16 ± 0.25 mm/s within the 0–0.5 mm/s speed range.

THE EXPERIMENTAL STUDY OF THE PREVENTIVE TREATMENT WITH VIBURNUM SARGENTII K. AND ELEUTHEROCOCCUS ON THE COMPOSITION OF FATTY ACIDS IN PHOSPHOLIPID COMPONENTS OF ERYTHROCYTE MEMBRANES EXPOSED TO INTOXICATION WITH NITRIC OXIDES

There are presented results of the study of the impact of intoxication with nitrogen oxides on to the fatty acids pattern of phosphatidylcholine and phosphatidylethanolamine of erythrocyte membrane in rats. The inhalation exposure with nitrogen oxides was carried out in inoculating chamber with the volume of 100 liters and designed according to the type of chambers by Kurliandsky B.A. with self-cleaning of contained air and regenerating system, specified temperature settings (20-22 °С) and the air humidity (40-60%). Intoxication by nitrogen oxides was performed for 6 minutes with a toxicant concentration of 4.3 mg/cu.m. (MAC in atmospheric air amount to 0.4 mg/m3). Water solutions of dry residue from viburnum extract “Kaliphen” and Eleutherococcus extract (alcohol was anticipatorily removed from preparations by evaporating in a vacuum) were administered daily through the tube intragastrically during 2 weeks in a dosage of 100 mg of total polyphenols per kg of body weight prior to intoxication by nitrogen oxides. The influence of nitrogen oxides was found to be accompanied by the elevation of the level of saturated fatty acids of all kinds and by the decline in unsaturated fatty acids content in the fatty acids pattern of phosphatidylcholine and phosphatidylethanolamine, the main structural phospholipids of biomembranes. The redistribution of fatty acids in erythrocyte membrane supposes the changes of its physicochemical properties, lability, and the complexity of the erythrocyte passage through the microvascular bed. Herbal polyphenol preparations Kaliphen and Eleutherococcus improved the survival rate of animals under the preventive administration before intoxication with nitrogen oxides. The introduction of extracts before intoxication is accompanied by the pronounced trend to the restoration of the ratio of saturated and unsaturated fatty acids in phospholipids of erythrocytes membranes. The preventive administration of Kaliphen before intoxication with nitrogen oxides was supposed to offer more pronounced protection of the fatty acids pattern of phospholipid fractions than those following the introduction of Eleutherococcus. The preventive administration of herbal extracts with polyphenol complexes in its contents under exposure to toxic substances is considered to be a the promising direction for research

THE EXPERIMENTAL STUDY OF THE PREVENTIVE TREATMENT WITH VIBURNUM SARGENTII K.AND ELEUTHEROCOCCUS ON THE COMPOSITION OF FATTY ACIDS IN PHOSPHOLIPID COMPONENTS OF ERYTHROCYTE MEMBRANES EXPOSED TO INTOXICATION WITH NITRIC OXIDES

There are presented results of the study of the impact of intoxication with nitrogen oxides on to the fatty acids pattern of phosphatidylcholine and phosphatidylethanolamine of erythrocyte membrane in rats. The inhalation exposure with nitrogen oxides was carried out in inoculating chamber with the volume of 100 liters and designed according to the type of chambers by Kurliandsky B.A. with self-cleaning of contained air and regenerating system, specified temperature setttings (20-220С) and the air humidity (40-60%). Intoxication by nitrogen oxides was performed during 6 minutes with a toxicant concentration of 4.3 mg/cu.m. (MAC in atmospheric air amount to 0.4 mg/m3). Water solutions of dry residue from viburnum extract “Kaliphen” and Eleutherococcus extract (alcohol was anticipatorily removed from preparations by evaporating in a vacuum) were administered daily through the tube intragastrically during 2 weeks in a dosage of 100 mg of total polyphenols per kg of body weight prior to intoxication by nitrogen oxides. The influence of nitrogen oxides was found to be accompanied by the elevation of the level of saturated fatty acids of all kinds and by the decline in unsaturated fatty acids content in the fatty acids pattern of phosphatidylcholine and phosphatidylethanolamine, the main structural phospholipids of biomembranes. The redistribution of fatty acids in erythrocyte membrane supposes the changes of its physicochemical properties, lability, and the complexity of the erythrocyte passage through the microvascular bed. Herbal polyphenol preparations Kaliphen and Eleutherococcus improved the survival rate of animals under the preventive administration before intoxication with nitrogen oxides. The introduction of extracts before intoxication is accompanied by the pronounced trend to the restoration of the ratio of saturated and unsaturated fatty acids in phospholipids of erythrocytes membranes. The preventive administration of Kaliphen before intoxication with nitrogen oxides was supposed to offer more pronounced protection of the fatty acids pattern of phospholipid fractions than those following the introduction of Eleutherococcus. The preventive administration of herbal extracts with polyphenol complexes in its contents under exposure to toxic substances is considered to be a the promising direction for research

Extracellular histones induce erythrocyte fragility and anemia

AbstractExtracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients.

Iodixanol, Constriction of Medullary Descending Vasa Recta, and Risk for Contrast Medium–induced Nephropathy

To determine whether a type of contrast medium (CM), iodixanol, modifies outer medullary descending vasa recta (DVR) vasoreactivity and nitric oxide (NO) production in isolated microperfused DVR. Animal handling conformed to the Animal Care Committee Guidelines of all participating institutions. Single specimens of DVR were isolated from rats and perfused with a buffered solution containing iodixanol. A concentration of 23 mg of iodine per milliliter was chosen to mimic that expected to be used in usual examinations in humans. Luminal diameter was determined by using video microscopy, and NO was measured by using fluorescent techniques. Iodixanol led to 52% reduction of DVR luminal diameter, a narrowing that might interfere with passage of erythrocytes in vivo. Vasoconstriction induced by angiotensin II was enhanced by iodixanol. Moreover, iodixanol decreased NO bioavailability by more than 82%. Use of 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (a superoxide dismutase mimetic) prevented both vasoconstriction with iodixanol alone and increased constriction with angiotensin II caused by CM. Iodixanol in doses typically used for coronary interventions constricts DVR, intensifies angiotensin II-induced constriction, and reduces bioavailability of NO. CM-induced nephropathy may be related to these events and scavenging of reactive oxygen species might exert a therapeutic benefit by preventing the adverse effects that a CM has on medullary perfusion.

Renal Alterations During Exercise

Proteinuria and hematuria are common during exercise. Proteinuria is usually due to glomerular or tubular changes or to an excessive production of protein as in myeloma. Certain clinical conditions can, however, result in a functional or temporary proteinuria, especially during pregnancy, fever, orthostasis, or following physical activity. Sport-related proteinuria following marching, exercise, and stress, was first observed in soldiers after long marching. Prevalence of proteinuria during exercise ranges from 18% up to 100% depending on type of exercise and its intensity. A higher incidence of proteinuria has been observed in some sports requiring great exercise intensity and it is certainly related to muscular work intensity and would decrease after prolonged training. Indeed, exercise-induced proteinuria is strictly related to exercise intensity rather than to exercise duration. Exercise aggravates the proteinuria of various nephropathies and that of renal transplant recipients. The prevalence of hematuria is higher in the athletic than the general population and the main difference is that sport-related hematuria resolves spontaneously after physical exercise while hematuria found in nonathletic population can be chronic. Sport-induced hematuria is influenced by exercise duration and intensity. Among the mechanisms underlying the exercise induced hematuria are increased body temperature, hemolysis, increased production of free radicals, and excessive release of catecholamines. Lactic acidosis, generated during anaerobic conditions, causes the passage of erythrocytes into the urine, through increased glomerular permeability.

A Novel Nucleotide Found in Human Erythrocytes, 4-Pyridone-3-carboxamide-1-β-d-ribonucleoside Triphosphate

We report the identification of a hitherto unknown nucleotide that is present in micromolar concentrations in the erythrocytes of healthy subjects and accumulates at levels comparable with the ATP concentration in erythrocytes of patients with chronic renal failure. The unknown nucleotide was isolated and identified by liquid chromatography with UV and tandem mass detection, (1)H nuclear magnetic resonance and infrared spectroscopy as 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate (4PYTP), a structure indicating association with metabolism of the oxidized nicotinamide compounds. Subsequently, we demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the chemically synthesized nucleoside precursor 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR). We noted preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP as well as a decrease in erythrocyte ATP concentration during incubation with 4PYR. Both the 4PYR phosphorylation and ATP depletion were blocked by an inhibitor of adenosine kinase. Plasma concentration of 4PYR was detectable but very low (0.013 +/- 0.006 microm) in contrast with the high daily urine excretion of this compound (26.7 +/- 18.2 micromol/24 h) in healthy subjects, indicating much greater renal clearance than other nicotinamide metabolites, nucleosides, or creatinine. We also noted a 40-fold increase in 4PYR plasma concentration in patients with chronic renal failure (0.563 +/- 0.321 microm). We suggest that 4PYTP formation in the erythrocytes is a hitherto unknown process aimed at sequestering potentially toxic 4PYR in a form that could be safely transported and subsequently released and excreted during passage of erythrocytes through the kidney.

Effect of oxygen consumption by measuring method on Po2transients associated with the passage of erythrocytes in capillaries of rat mesentery

Mathematical models have predicted the existence of Po(2) gradients between erythrocytes in capillaries in the usual case where plasma contributes substantial resistance to oxygen diffusion. According to theoretical predictions, these gradients could be detected as rapid Po(2) fluctuations (erythrocyte-associated transients, EATs) along the capillary. However, verification of a model and correct choice of its parameters can be made only on the basis of direct experimental measurements. We used phosphorescence quenching microscopy to measure Po(2) in 52 capillaries of rat mesentery to obtain plasma Po(2) values 100 times/s at a given point along a capillary. A 532-nm laser generated 10-mus pulses of light, concentrated by a x100 objective, onto a spot 0.9 mum in diameter. The presence of erythrocytes in the excitation region was detected on the basis of phosphorescence amplitude (PA), proportional to the amount of plasma encountered by the laser beam, and on the basis of the intensity of transmitted laser light (LT), detected by a photodiode placed under the capillary. The data revealed correlated waveforms in PA, LT, and Po(2) in capillaries. The magnitude of the Po(2) gradients between erythrocytes and plasma was correlated with average capillary Po(2). EATs in Po(2) were more readily detected in capillaries with relatively low oxygenation. The correlation coefficients between PA and Po(2) for the half of the capillaries (n = 26) below the median Po(2) (mean Po(2) = 17 mmHg; R = -0.72) was higher than that for the other half (mean Po(2) = 39 mmHg; R = -0.38). These results support the theoretical predictions of EATs and plasma Po(2) gradients in capillaries.

Effects of alpha 1-acid glycoprotein on erythrocyte deformability and membrane stabilization.

The effects of alpha(1)-acid glycoprotein (AGP) on human erythrocyte membrane were examined in vitro. Bovine and dog AGP, in addition to human AGP or asialo human AGP were used, and the collected data were compared with that for human serum albumin (HSA). A new technique developed by Kikuchi was used to investigate erythrocyte deformability. The addition of AGPs including human AGP facilitated the passage of human erythrocytes with an average diameter of 7.2 microm suspended in phosphate buffered saline (PBS) through a 5 microm wide microchannel; hemolysis was suppressed after the passage. The stabilizing effects of AGPs on membrane were evaluated. Human AGP prevented hemolysis induced by hypotonic phosphate buffer solution. The effects of human AGP on the oxidative changes in erythrocytes exposed to oxygen radicals were investigated. Human AGP protected erythrocytes from H(2)O(2) and prevented the oxidation of dihydrorhodamine 123 to rhodamine 123 from H(2)O(2). We propose that the antioxidant activity of human AGP is due to the binding of free radicals. In all studies, the effects of human AGP on erythrocytes might not be a function of the negative charge associated with sialyl residues, because the presence of N-acetylneuraminic acid had no effect. However, human AGP may promote microcirculation and antioxidant activity compared with HSA. No species differences in the physiological function of AGP were found. These results suggest that an increase in the AGP content of serum above the normal value found under pathological conditions facilitates the passage of erythrocytes through capillaries, stabilizes erythrocyte membranes and protects against oxidative stress, all of which are favorable for microcirculation.

Role of leukotrienes in stress-induced damage to the heart.

Experiments on rabbits with epinephrine-induced damage to the heart showed that progression of necrotic degenerative processes in the myocardium is augmented by increased content of prostaglandin F(2alpha)and predominance of constrictive over vasodilatory effects in the E and F(2alpha)prostaglandin system. Twenty-four hours after injection of epinephrine we observed an increase in blood concentrations of myofibrillar fraction of creatine phosphokinase, serotonin and histamine, and the F(2alpha)/E prostaglandin ratio. The concentration of leukotriene B(4)increased during the urgent adaptation period, which correlated with a decrease in elastic properties of erythrocytes. In vitro passage of erythrocytes through a 2.5-micro capillary sieve sharply decreased, which played an important role in the progression of myocardial and cerebral hypoxia and ischemia. Myocardial necrosis and endothelial dysfunction developed 24 h later aggravate these pathological shifts.

β‐Adrenergic agonists regulate cell membrane fluctuations of human erythrocytes

1. Mechanical fluctuations of the cell membrane (CMFs) in human erythrocytes reflect the bending deformability of the membrane-skeleton complex. These fluctuations were monitored by time-dependent light scattering from a small area ( approximately 0. 25 microm2) of the cell surface by a method based on point dark field microscopy. 2. Exposure of red blood cells (RBCs) to adrenaline (epinephrine) and isoproterenol (isoprenaline) resulted in up to a 45 % increase in the maximal fluctuation amplitude and up to a 35 % increase in the half-width of the amplitude distribution. The power spectra of membrane fluctuations of control and treated cells revealed that adrenaline stimulated only the low frequency component (0.3-3 Hz). Analysis of the dose-response curves of beta-adrenergic agonists yielded an EC50 of 5 x 10-9 and 1 x 10-11 M for adrenaline and isoproterenol, respectively. Propranolol had an inhibitory effect on the stimulatory effect of isoproterenol. These findings show a potency order of propranolol > isoproterenol > adrenaline. 3. The stimulatory effect of adrenaline was a temporal one, reaching its maximal level after 20-30 min but being abolished after 60 min. However, in the presence of 3-isobutyl-1-methylxanthine, a partial stimulatory effect was maintained even after 60 min. Pentoxifylline and 8-bromo-cAMP elevated CMFs. However, exposure of ATP-depleted erythrocytes to adrenaline or 8-bromo-cAMP did not yield any elevation in CMFs. These findings suggest that the beta-agonist effect on CMFs is transduced via a cAMP-dependent pathway. 4. Deoxygenation decreased CMFs and filterability of erythrocytes by approximately 30 %. The stimulatory effect of isoproterenol on CMFs was 2.2-fold higher in deoxygenated RBCs than in oxygenated cells. 5. Exposure of RBCs to adrenaline resulted in a concentration-dependent increase in RBC filterability, demonstrating a linear relationship between CMFs and filterability, under the same exposure conditions to adrenaline. These findings suggest that beta-adrenergic agonists may improve passage of erythrocytes through microvasculature, enhancing oxygen delivery to tissues, especially under situations of reduced oxygen tension for periods longer than 20 min.

Pentoxifylline Improves Skeletal Muscle Recovery After Acute Ischemic and Reperfusion Injury

Purpose: Skeletal muscle ischemia is associated with complex interactions involving neutrophils, erythrocytes, oxygen free radicals, and complement factors that lead to development of reperfusion injury on restoration of blood flow. Pentoxifylline is known to decrease neutrophil margination, improve erythrocyte passage through the capillary system, and possibly serve as an oxygen radical scavenger. The authors hypothesized that the addition of pentoxifylline to the reperfusate might improve muscle recovery after acute ischemic injury. Methods: Twenty-four New Zealand White rabbits underwent vascular isolation of both hind limbs, leaving the external iliac and femoral arteries as sole inflow arteries for each respective limb. The animal was secured to an operating board such that each tibialis anterior muscle was attached to a force transducer, and muscle contractions after maximal tetanic stimulation were recorded on an oscilloscope at timed intervals. Acute occlusion was induced in the right hind limb by use of removable vascular clips and continued until < 15% of contralateral limb function was achieved. The animal was then (continued on next page) (Abstract continued) reperfused with saline "control " or pentoxifylline (50 mg/kg) "treated." Muscle function was recorded in each limb at 5, 30, 60, 90, and 120 minutes after reperfusion. Results: A continual trend of muscle function improvement was noted in the pentoxi fylline-treated group, reaching statistical significance compared with controls after 30 minutes (16.8% of contralateral limb; P<0.04) and 120 minutes (36.7% of contralat eral limb ; P<0.04) of reperfusion. Control animals failed to demonstrate significant improvement in muscle function over the 2-hour reperfusion period. Conclusion: Pentoxifylline improved muscle function recovery after acute ischemic injury in the rabbit hind limb model.

Red cell filterability determined using the cell transit time analyzer (CTTA): effects of ATP depletion and changes in calcium concentration

Cell transit time analysis (CTTA) is a new filtrometric technique for assessing red blood cell deformability by measuring the conductivity change caused by passage of erythrocytes through a polycarbonate filter. Most reported studies to date using CTTA have focused on the transit time (TT), the duration of passage of an individual red cell through a micropore. Bulk flow rate has not been previously measured via CTTA. The use of new enzyme based cleaning solutions make it possible to reduce clogging in micropore filters. Therefore, valid measures of the number of red cell transits per unit time (counts/s: C/S) can now be obtained. We evaluated both parameters. TT and C/S, as indicators of red cell filterability. Our goal was to evaluate the effect of metabolic changes shown by alternative techniques to affect red cell deformability. The two best established factors are changes in intracellular [ATP] and [Ca 2+]. ATP depletion produces a very small increase in TT but a very marked decrease in C/S. In contrast, the addition of low concentrations of calcium produces an increase in TT with minimal decrease in C/S. The effects of calcium appear to be complex. The substantial changes in intracellular calcium induced by the ionophore A23187 result in a curvilinear pattern of increase in transit times and reduction in counts per s. Lanthanum, which inhibits egress of intracellular calcium, causes an increase in TT with a drop in C/S. We conclude that CTTA demonstrates the same changes in red cell deformability measurable by alternative filtrometric techniques; however, CTTA furnishes two separate and independent parameters which may be used to evaluate red cell deformability.

In vivo neutrophil sequestration within lungs of humans is determined by in vitro "filterability"

Neutrophils are normally delayed in transit through the lung microcirculation, relative to the passage of erythrocytes. This sequestration contributes to a pulmonary pool of neutrophils that may relate to the relative inability of neutrophils to deform compared with erythrocytes when in transit in the pulmonary capillaries. A micropore membrane was used to model the human pulmonary microcirculation, in which cell deformability was measured as the pressure developed during filtration of the cells through the membrane at a constant flow. We demonstrated a significant correlation between in vitro deformability and in vivo lung sequestration of indium-111-labeled neutrophils in 10 normal subjects (r = 0.69, P less than 0.02). In eight patients with stable chronic obstructive pulmonary disease, this relationship was not significant (r = -0.2, P greater than 0.05). Furthermore, in a subject with microscopic pulmonary telangiectasia known to allow significant passage of 30-microns microspheres, neutrophils passed through the lungs without delay. Moreover, neutrophils from patients studied acutely with an exacerbation of chronic obstructive pulmonary disease were temporarily less deformable (P less than 0.01). These studies confirm that cell deformability is an important determinant of the normal neutrophil sequestration within the lungs. Changes in cell deformability may alter the extent of this sequestration.

The time-space correlation method for measurement of erythrocyte velocity in microvessels using a CCD linear image sensor

A new method for measuring erythrocyte velocity in the microvascular system was developed. A correlation function varying both time and distance, “time-space correlation method,” was used in order to improve the time correlation and the space correlation methods. A CCD linear image sensor installed at the center of the focal plane of a camera was used to convert the passage of erythrocytes through the microvascular system to electrical signals via intravital microscopy. The method was applied to small vessels in the mesentery of rats. Using the present method, the time required for the measurement and the length of the vessel necessary for the measurement have been reduced, as compared with other methods.

Endothelial nuclear bulging: Morphological evidence for an intraglomerular perfusion regulating mechanism in the axolotl

Kidneys of the Axolotl ( Ambystoma mexicanum: Urodela, Amphibia) have been investigated by light and electron microscopy as well as microvascular corrosion casting. Numerous glomerular endothelial nuclei bulge into the glomerular capillary lumen. It is concluded that these cells virtually stop the blood-flow in the respective vessel by blockage of erythrocyte passage. A proposal for an intraglomerular regulation mechanism of capillary perfusion (and thus filtration) by endothelial isometric contraction in the Axolotl is made on the basis of morphological methods.

Microvascular responses in rabbit skeletal muscle after fixed volume hemorrhage

The effect of hemorrhage on the microvascular responses in the tenuissimus muscle was studied by means of intravital microscopy in rabbits anesthetized with urethan. The rabbits were bled 30% of their calculated blood volume within 3 min. Hemorrhage initially caused mean arterial pressure to drop from 70 +/- 7 to 26 +/- 5 mmHg. During the subsequent 30-min observation period it increased to 43 +/- 8 mmHg. The transverse arterioles (TRs), supplying both muscle tissue proper and adjacent connective tissue, gradually constricted to 75% of control over the 30-min period. Terminal arterioles (TEs) branching from the TR in the muscle tissue constricted to 65% in 10 min and then gradually relaxed, eventually reaching 80% of control diameter. The constriction of the TEs was confined to a short sphincterlike structure (10-20 microns) at the origin of the bifurcation. Upon constriction, the diameter of the sphincterlike structure was less than the critical diameter for erythrocyte passage. Given that the effective blood viscosity in the narrow TE is strongly dependent on luminal diameter, the overall effect on blood flow and its distribution in the tenuissimus muscle was a dramatic reduction of volume flow to 20-30% of the control value. During the early phase, the reduced flow was diverted to the connective tissue at the expense of nutrient flow to the muscle tissue. This early blood flow pattern gradually reversed, partially restoring nutrient flow to the muscle fibers.

Dynamic Change of the Vascular Wall from Transmural to Intercellular Passage of Red Blood Cells Observed in Splenic Regeneration

Rat splenic tissues were autotransplanted into the major omentum, and the operated animals were treated with phenylhydrazine to investigate the passage of erythrocytes through the vascular wall during splenic regeneration. Both ways of the passage were differentiated during regeneration. From day 1 to day 5 after transplantation, the pores were formed in the endothelial cells, through which erythrocytes (chiefly reticulocytes) migrated, and were closed by the basal lamina when erythrocytes did not penetrate. From day 7 to day 10, endothelial cells proliferated, and some of them were transformed into sinus endothelial cells containing condensed microfilaments and formed the interendothelial slit, but few erythrocytes passed there at this stage yet. On and after day 11, when the sinus endothelial cells exhibited well-developed microfilaments, reticular cells contained moderately developed microfilaments and the basal lamina developed well, the slits were opened, where a large number of erythrocytes passed. These results showed, concerning the passage of blood cells, that the vascular wall in the splenic autografts changed from the transmural pattern to the intercellular one after a marked proliferation of endothelial cells and that the effective passage of erythrocytes was closely associated with the development of microfilaments in the cytoplasm of endothelial cells and basal lamina as well as the interaction of reticular cells.

In vitro generation of dysmorphic erythrocytes

In hematuria, dysmorphic red blood cells found in the urine sediment are known to indicate glomerular origin of bleeding. To better understand the mechanisms which might cause the typical membrane changes in dysmorphic erythrocytes in vivo, we have exposed normal human erythrocytes in vitro to an osmotic and enzymatic environment similar to that of the nephron. In addition, treated or untreated erythrocytes were exposed to a hemolytic environment. The morphology of the cells was evaluated by light and electron microscopy in comparison of that of dysmorphic erythrocytes obtained in vivo. Neither the passage of erythrocytes in sequence through various solutions each simulating the tubule fluid in a different part of the nephron, nor exposure to urinary or renal enzymes or to serum per se causes the typical membrane alterations. Upon exposure of passaged erythrocytes to a hemolytic environment, however, 50 to 90% of the cells become dysmorphic in a time and dose dependent fashion. By light and electron microscopy these dysmorphic erythrocytes obtained in vitro are indistinguishable from those obtained in vivo. In contrast, non-passaged red blood cells are not affected by the same hemolytic environment. We conclude that osmotically challenged erythrocytes exposed to a hemolytic environment develop dysmorphic membrane alterations.