Biorheology

Water and secretions interact in airways to produce the sol and gel layers that allow for entrapment of foreign materials and subsequent clearance by ciliary movement and by cough. Active Cl ion transport produces fluid, and this process is activated by products of mast cells (leukotrienes), eosinophils (major basic protein), and by other inflammatory mediators (prostaglandins, bradykinin). Gland secretions produce the bulk of the volume of secretions. Airway irritation stimulates gland secretion reflexly via vagal muscarinic pathways. Recently, the sensory nerves have been discovered to release substance P and other neuropeptides when the airways are irritated. The stimulatory effects of neuropeptides on gland secretion (and on other inflammatory sites) are modulated by enkephalinase, a membrane-bound enzyme that cleaves neuropeptides and thereby inactivates them. Up- or down-regulation of enkephalinase is predicted to change the degree of inflammatory response to neuropeptides. Finally, the cell surface of airway epithelial cells have been discovered to secrete large molecular weight glycoconjugates; these secreted products are increased markedly by a series of proteinases produced by inflammatory cells (neutrophils, mast cells) and by bacteria. Their exact physiologic roles are still unknown but they may contribute to the bulk and viscoelastic properties of airway secretions, and they may serve an important role in bacterial, viral and inflammatory cell adhesion.

Airway epithelia possess transepithelial ion transport processes which may help to regulate the fluid content of airway secretions. Chloride secretion promotes fluid movement from blood to airway lumen. Active absorption of Na favours fluid movement in the opposite direction. The balance between these two processes can be altered by a number of agents which stimulate Cl secretion. The importance of ion transport for normal mucociliary clearance is suggested by the finding that airway epithelia in patients with cystic fibrosis are unable to secrete chloride. This defect may cause the characteristically sticky and tenacious mucous secretions which are the major cause of death in this disease.

Respiratory mucus and mucosa possess highly hydrophilic structures which are difficult to preserve using standard fixative methods. The close interaction between cilia and mucus can be observed after instantaneously interrupting the ciliary movement using ultra rapid and cryosubstitution fixation methods. Mucus possess several rheological properties such as pseudoplasticity, elastothixotropy, spinability and adhesiveness. Rheological properties of mucus may control, per se, the ciliary beating frequency. By measuring the mucociliary frequency on the excised mucus-depleted frog palate of native mucus and xanthan gum using a simulant of mucus, we observed that beyond an optimal value of viscosity (close to 12 Pa·s) the mucociliary frequency and transport rate decrease in parallel. Other rheological factors such as adhesion and spinability of mucus can also be implicated in the regulation of the mucociliary transport rate.

The interposition of a neutral starch gel greatly retarded bulk ionic movement by free flow. A mucus (charged) gel preparation of identical concentration and thickness further retarded ionic diffusion. The findings suggest that the charges in the mucus matrix may exert an ionic exclusion effect (Donnan Exclusion). thus retarding other ionic diffusion. We speculate that a mucus layer may physiologically behave as an ion exchange gel column and modify the traffic of charged ions through it.

The mucus secreted by the body surface of the terrestrial pulmonate slug, Ariolimax columbianus, is a variable mix of products of the giant mucous and channel cells of the skin. An in vitro sac preparation allows the study of control and products of these two cell types. Mucins in membrane-bound granules are released by the mucous cells on mechanical or electrical stimulation of the skin. The product of the channel cells is a blood ultrafiltrate, modified by reabsorption of Na⁺ and Cl⁻ ions and transfer into the fluid of K⁺ and HCO₃ ⁻ ions (processes of secretion inhibited by ouabain, amiloride and furosemide; and by other epithelial blocking agents). Rates of secretion can be increased or decreased by gastropod neurotransmitters (acetylcholine, serotonin, and others), and by prostaglandin E₂ and indomethacin.

Several noninvasive light scattering techniques are reviewed that can be utilized in rheological studies of mucus and other soft biological matrices. Included are quasielastic light scattering techniques to determine the compressibility moduli of polymer lattices, resonance methods that can be used to measure the shear modulus of weak gels, assays that probe the swelling and contraction of mucin aggregates, and microscope based light scattering techniques to probe gelation within single cells.

The glycoconjugate composition of tracheal secretions varies with physiological and pathophysiological parameters. Believing that these differences might be explained by metabolic or regulatory modifications of particular cell types, we have developed strategies for biochemical analysis at the cellular level. We have produced monoclonal antibodies whose determinants arc restricted to a single secretory cell type (serous, mucous, or goblet cell granules, or ciliated cell glycocalyx). By enzyme immunoassay (ELISA), we have characterized four of the antibodies biochemically, and have also used the antibodies as quantitative molecular probes to detect release of antigen from mixed cell explants. Four of the antigens are carried by carbohydrate moieties of high molecular weight glycoproteins. Western blot analysis shows their molecular weight in reducing gels (SDS-PAGE) to exceed 200 kD. When used in parallel with pulse-chase labeling studies, the antibodies are both more sensitive and specific (than bound radioactivity) in detecting gland or goblet cell secretion in response to autonomic drugs or proteases. We have also isolated and cultured serous gland cells for physiological and biochemical studies. These cells express serous cell phenotype as reflected by ultrastructure, histochemistry, and lysozyme activity. Biochemical analysis of their secretory products reveals glycoconjugate components which are heterogeneous with respect to both molecular weight and charge. Radiolabeled secretory products eluting in the void volume of Sepharose Cl 4B were completely degraded by chondroitinase ABC. This indicates that the major glycoconjugate produced by serous cell is a proteoglycan resembling chondroitin sulfate.

The relationships between mucus rheology, depth of mucus layer and clearance by simulated cough were examined in a study employing a model plexiglass trachea lined with gels formed from locust bean gum crosslinked with sodium tetraborate. The viscoelastic properties of the mucus simulants were determined by magnetic rheometry at 100 rad/s and expressed as mechanical impedance (dynamic stress/strain ratio) and loss tangent. Cough was simulated by opening a solenoid valve connecting the model trachea to a pressurized tank, using an upstream flow-constrictive clement to shape the flow profile to approximate the pattern seen in a normal adult. Mucus clearance was quantitated by observing the movement of contrasting marker particles placed in the mucus layer. The median particle displacement was defined as the clearance index, Cl. For any initial depth of mucus, Cl increased with driving pressure in the tank, and for a given driving pressure, Cl increased linearly with increasing mucus depth. For a given driving pressure and depth, Cl decreased with increasing mechanical impedance of the mucus. At constant mechanical impedance, Cl increased with increasing loss tangent, in other words, cough clearance was impeded more by elasticity than viscosity. Mucus clearance was associated with transient wave formation in the lining layer. Thus dependence on viscoelasticity is consistent with observations that airflow-mucus interaction and wave formation are impeded by elasticity. The clearance vs. loss tangent relationship for cough is opposite to that found for ciliary clearance (Biorheology 1980, 17, 249), suggesting a natural balance in viscosity and elasticity for mucus to be cleared by both mechanisms. The differences in clearance-viscoelasticity relationship also suggest that rheological adaptation of mucus should occur when the mode of clearance shifts from ciliary action to cough.

Recently a computerized electro-optical method was developed which enables one to simultaneously measure the frequency and the wavelength of the metachronal waves in beating cilia. The method is based on measurement of scattered light from two areas at a given distance apart. The distance between measured areas can be varied from zero to hundreds of microns. The relative ease of the measurement and data analysis of this method enable one to create large statistical ensembles in order to obtain reliable averages.

In this work we show that in addition to the previously mentioned parameters this system can measure directly the velocity of the metachronal wave. It was found that the average velocity in the tissue culture from frog’s palate epithelium at room temperature is ∼ 270 μm/sec, about twice the average particle velocity at the frog’s palate.

An approximately 500,000Da subunit characterizes the structural glycoprotein of mucus. This unit is composed of a rod-like very heavily glycosylated protein chain with an unglycosylated cysteine rich region at its end. It is proposed that the ‘bare’ peptide portion of the subunit forms itself into a lectin by undergoing a disulfide bond stabilised conformational fold. The lectin binding site, R, is assumed to be selective for a rare sugar sequence, which, when present, creates a binding site R*. The site R* has to be relatively rare. The average number <s*> of sidechains which contain R* per subunit will only be of order 1. When <s*> ⩽ 1 reasonably long, chain-like aggregates are formed which behave like coiling polymer chains with the subunit the Kuhn statistical element. When <s*> > 1 the entire system forms one structure. Data obtained from the literature are analysed. They favor finite size, separate chains. The Kuhn statistical element length, derived from the data, is shown to agree well with the lectin model hypothesis.

Mechanical spectroscopy has been used to study the structure of mucus gel taken from the surface of the pig gastrointestinal tract. Mucus from stomach, duodenum and colon was insoluble and its mechanical properties, characteristic of a weak viscoelastic gel, were unchanged in saline, acid (pH 2) and denaturants. Small intestinal mucus gel which was of poorer quality, was disrupted following exposure to acid and denaturants. Concentration of purified glycoprotein produced gels that had mechanical spectra with the same profiles as the respective native secretion except for reconstituted small intestinal mucus which was of better quality and similar to the other native and reconstituted gels. Reduction of S-S linkages or proteolysis of all mucus gels caused a collapse of structure to give profiles typical of a viscous solution. This collapse of gel structure was shown to result from a breakdown of the covalent polymeric structure of the component glycoproteins. A linear correlation for mucus gels was observed between gel quality (as defined by tan δ) and the ratio of polymeric glycoprotein to its degraded lower molecular weight subunit. Human gastric mucus from a histologically normal stomach also had the characteristics of a weak viscoelastic gel, although that from patients with peptic ulcer disease has a significantly reduced content of polymeric glycoprotein.

Mucin-containing granules, produced by mammalian goblet cells in vitro, undergo massive post-exocytotic swelling (23). Their swelling kinetics are similar to the swelling of condensed artificial polymer gels (22). Earlier, we proposed that mucins are condensed in the secretory granule and expand by swelling during or after exocytosis (21). The swelling of mucus is affected by ionic influences, as it is governed by a Donnan equilibrium process (21). However, the effect of cations on the swelling of newly released mucins had not yet been investigated. Calcium has been found in high concentration inside secretory granules of mucin-secreting cells (18, 9, 25), and is also elevated in the mucus of cystic fibrosis patients (17). The present experiments were designed to study the effect of extracellular Ca⁺⁺ concentration on the swelling kinetics of the newly released secretory product of respiratory goblet cells in vitro. The data show that extracellular Ca⁺⁺, in concentrations similar to those found in the mucus of cystic fibrosis patients (2 to 4 mM) can produce a four-fold decrease in the diffusivity of the newly released mucin polymer network, resulting in a slow rate of swelling, and a mucus that remains thick for long periods of time. The present findings are in agreement with the Donnan equilibrium hypothesis for the regulation of mucus swelling and rheology (21), and bear important implications for the pathophysiology of cystic fibrosis.

Observations of flow velocity profiles over frog mucociliated palate are used to estimate viscosity, shear rate and shear stress in the periciliary flow field. The ability of cilia to generate significant shear stress at long distances and their utility as rheometers are examined. It is proposed that the depth of significant ciliary shear penetration into the periciliary fluid is sufficient to move mucus masses well beyond the ciliary tips, obviating the need for tip penetration where anchoring phenomena are sufficiently reduced.

Gelsolin is a Ca²⁺-binding protein of mammalian leukocytes, platelets and other cells which has multiple and closely regulated powerful effects on actin. In the presence of micromolar Ca²⁺, gelsolin severs actin filaments, causing profound changes in the consistency of actin polymer networks. A variant of gelsolin containing a 25-amino acid extension at the NH₂-terminus is present in plasma where it may be involved in the clearance of actin filaments released during tissue damage. Gelsolin has two sites which bind actin cooperatively. These sites have been localized using proteolytic cleavage and monoclonal antibody mapping techniques. The NH₂-terminal half of the molecule contains a Ca²⁺-insensitive actin severing domain while the COOH-terminal half contains a Ca²⁺-sensitive actin binding domain which does not sever filaments. These data suggest that the NH₂-terminal severing domain in intact gelsolin is influenced by the Ca²⁺-regulated COOH-terminal half of the molecule. The primary structure of gelsolin, deduced from human plasma gelsolin cDNA clones, supports the existence of actin binding domains and suggests that these may have arisen from a gene duplication event, and diverged subsequently to adopt their respective unique functions. The plasma and cytoplasmic forms of gelsolin are encoded by a single gene, and preliminary results indicate that separate mRNAs code for the two forms. Further application of molecular biological techniques will allow exploration into the structural basis for the multi functionality of gelsolin, as well as the molecular basis for the genesis of the cytoplasmic and secreted forms of gelsolin.

The interaction of platelets with natural and artificial surfaces is briefly reviewed, emphasizing the role of the platelet glycoprotein Ib and IIb/IIIa receptors. Studies utilizing monoclonal antibodies to these receptors for the diagnosis and therapy of hemorrhagic and thrombotic disorders are described, indicating the potential of such agents as platelet inhibitors.

This presentation is aimed at giving some background information on molecular biology, thus serving as an introduction to the symposium on Molecular Biorheology held during the sixth International Congress of Biorheology in Vancouver. The papers presented at this Symposium indicate that the use of molecular biological techniques allows the understanding of normal and abnormal rheological properties of cells and organs at the molecular level. It is hoped that these examples will provide an impetus for us to open new frontiers of research in biorheology by taking advantage of the powerful tools developed from recent advances in molecular biology.

The application of molecular biology to the study of the human erythrocyte membrane is presented in this report. We describe the strategy employed to clone DNA sequences encoding the membrane skeletal protein, protein 4.1. We demonstrate how these sequences may be utilized to deduce detailed structural and functional information about the 4.1 polypeptide. We also illustrate the use of cloned 4.1 DNA sequences as probes to explore the structural organization of the protein 4.1 gene in normal individuals and in patients with dysfunctional erythrocyte membranes. Ultimately it will be possible to generate a molecular description both of the structural proteins which constitute the membrane skeleton, and of the genetic mechanisms regulating their expression in erythroid and nonerythroid cells.

The flow velocities in glass and silastic lateral aneurysm models were quantitatively measured with the non-invasive laser Doppler method. The influences of the elasticity of the wall, the pulse wave and the properties of the perfusion medium on the intra-aneurysmal circulation were investigated. As shown previously, the inflow into the aneurysm arose from the downstream lip and was directed toward the center of the fundus. Backflow to the parent vessel took place along the walls of the fundus. with non-pulsatile perfusion, flow velocities in the center of the standardized aneurysms varied between 0.4 and 2% of the maximum velocity in the parent vessel. with pulsatile perfusion, flow velocities in the center of the fundus ranged between 8 and 13% of the flow velocity in the axis of the parent vessel. Flow velocities in the aneurysms were slower with a polymer suspension with blood-like properties compared to a glycerol/water solution. Flow velocity measurements near the aneurysmal wall allowed the estimation of the shear stresses at critical locations. The maximum shear stresses at the downstream lip of the aneurysm were in the range of the stresses measured at the flow divider of an arterial bifurcation. The present results suggest that in human saccular aneurysms intra-aneurysmal flow and shear stress on the wall are directly related to the pulsatility of perfusion, i.e. the systolic/diastolic pressure difference and that the tendency to spontaneous thrombosis depends on the viscoelastic properties of the blood, namely the hematocrit.

A phase-by-phase analysis of local flow patterns at the venous anastomosis of an arteriovenous hemodialysis angioaccess loop graft (AVLG) was made. The study was carried out in an elastic, transparent Silastic in vitro flow model, which duplicates the detail geometry of the AVLG obtained from an animal model (30+ kg dogs with 12 weeks bilateral femoral AVLG implantation). The flow model was installed in a mock pulsatile flow loop system designed to simulate physiological conditions. Flow visualization was made in laser-illuminated flow fields using a high-speed cine camera. Analysis of the high-speed cine indicates there is a distinct separation region downstream of the anastomotic toe in the median plane and a stagnation region that oscillates along the opposite wall. During inward motion of the vessel wall, accumulation of particles in the separation region and the nearby stagnation region is observed. A large swirl appears in the distal vein during end-systolic period. A double-helical flow pattern occurs further down in the distal vein. Retrograde flow in the distal vein occurs in an “oscillating” manner following each cardiac cycle.

The phasic velocity field in the vicinity of the venous anastomosis In a hemodialysis angioaccess arteriovenous fistula loop graft (AVLG) is investigated employing a laser Doppler anemometer (LDA) system. Detailed LDA velocity profiles are obtained by sectional survey performed in a transparent, elastic flow model which was fabricated to represent the geometry of the AVLG system under physiological pressure and flow waveforms. The geometry of the flow model was based on a silicone rubber cast obtained from an experimental dog model. In the present study, detailed distribution of velocity profiles is obtained. The distribution of wall shear stress in the model is computed from the slope of the local velocity profiles near the wall. The relationship between the results obtained by flow visualization and the LDA measurement is discussed.

The optimal geometry of the vascular bifurcation is interpreted on the basis of the principle of minimum work. We consider the energy expenditure due to the viscosity of blood, and that for maintaining the metabolic states of the blood cells and of the vessel wall. It is shown that the optimal radii of the stern and branch vessels and the optimal branching angle are related to two parameters which represent the morphologic and metabolic states of the blood and the vessel wall. In the special case of symmetrical bifurcation, it was found that as the metabolic demand of the vessel wall becomes more apparent when compared with that of the blood, the branch radius relative to that of the stern takes values of from 0.794 down to 0.758 minimally, and the angle from 37.5° up to 48.7° maximally with respect to the direction of the stern.

An electrochemical surface shear stress measurement was applied to a model of unilateral arterial stenosis. The unilateral stenosis model was made up of a removable stenosis plug, in an electrochemical shear stress measurement test section with 100 cathodes. Three dimensional wall shear stress distribution was measured under steady flow field. At a relatively low Reynolds number, Re=270, there was a characteristic high and low wall shear distribution pattern downstream of the unilateral stenosis. There were also remarkable high shear stress areas on the opposite wall up- and downstream, and both side walls of the stenosis upstream. It was clearly shown that detailed three dimensional structure of the flow field must be studied in order to correlate it to pathological findings.

A dilute suspension of Spirulina Microalgae is found to exhibit radial migration in laminar flow in a 650 micron vertical tube. As the tube Reynolds number increases, the particles concentrate in a narrower region around the tube axis. When the turbulent regime is approached, the particles disperse as expected.

Three groups of male Sprague Dawley rats received methimazole without or with Na-thyroxine in drinking water (3 and 0.33 mg T₄/l, respectively) to induce characteristic alterations of their thyroid status (hypothyroid, hyperthyroid, euthyroid). A fourth group served as an untreated control without any additive to the drinking water. With respect to the different thyroid status, the following changes in the blood parameters were found: increasing plasma-T₃-levels caused a reduction in plasma viscosity, in total plasma protein and in α 1 -globulin, but an increase in hematocrit, whole blood viscosity, the number of erythrocytes and leukocytes, α 2 -globulin and β-globulin. It was concluded that the increase in the plasma viscosity in the hypothyroid status is mainly due to an alteration of the plasma protein pattern, and that the increase in whole blood viscosity in the hyperthyroid rat is a consequence of increased hematocrit.

In an effort to find a method to intensify the rheological destruction of tumor cells, rat fibrosarcoma KMT 17 cells in ascitic form were exposed to rheological stresses in test solutions; modified Eagle medium with EDTA, and phosphate buffer solution without divalent cations or with verapamil. KMT 17 cells were exposed to a uniform shear stress produced by the rotation of a rotating cone plate viscometer for 1 to 2 hours and to the strong deformation by the passage through Nuclepores of 10, 8 and 5 micrometer. KMT 17 cells suspended in the test solutions were more effectively destroyed by the stresses than those suspended in normal solutions containing divalent cations without any other calcium-suppressing agents. These results suggest that the elimination of divalent cations and/or the block of calcium ion channels of cell membranes intensify the rheological destruction of tumor cells migrating in the circulatory system.

The influence on fluid flow of the fixed charge on the surface of capillaries is calculated using the linearised Poisson-Boltzmann equations. The results depend strongly upon the ratio of the capillary radius to the Debye length. At physiological ionic strength, the Debye length is less than 1 nm and electrostatic effects are negligible. In particular, they can not explain the Copley-Scott Blair phenomenon in artificial capillaries. Electrostatic effects can be significant in smaller channels and it is calculated that in intercellular clefts in the capillary endothelium the apparent viscosity of the fluid may increase more than 50%. These effects can also be important in the flow in the narrow gap between a red cell and the blood capillary wall. Using the Fitzgerald-Lighthill model of this flow and parameters typical of the human microcirculation, the theory predicts that the apparent viscosity in the gap will be increased by about 5%.

The steady state non-Newtonian viscosity of whole human blood has been widely studied as a function of the shear rate; and used to characterize the blood in various pathological disorders. In our previous studies, we demonstrated that blood is a thixotropic fluid. Its time-dependency and shear rate dependency of rheological behavior can be represented by an equation developed by Huang. Parameters of the equation can be used for the characterization of an individual’s blood. They provide information, such as the kinetic rate constant of breakdown of RBC rouleaux to individual erythrocytes and the relative amount of rouleau formation in the dynamic equilibrium between rouleaux and individual erythrocytes. In this communication, the thixotropic parameters from blood samples of fifteen apparently healthy human subjects were investigated. When compared to the use of apparent viscosity values for the correlation with a pathological disorder, thixotropic parameters are preferable. The mean values of thixotropic parameters obtained from apparently healthy human subjects provide a base for comparison with the same parameters as obtained from blood samples of patients with certain pathological disorders involving the circulation.

The rheological properties of whole human blood exhibit thixotropic behavior at low shear rates up to about ten reciprocal seconds (1). The accepted cause of this shear rate-dependent and time-dependent behavior is the progressive breakdown of rouleaux into individual red cells. Huang developed a rheological equation which incorporates the kinetics of rouleau breakdown in his models (2). This five-parameter equation was used successfully to represent the hysteresis loop and the torque-decay curve of whole human blood. Numerical values of these five thixotropic parameters, which characterize the rheological behavior of the blood from apparently healthy human subjects, were established (3). In this communication, we examine the effect of hematocrit on each of the above mentioned parameters. The results show that the following parameters will increase their values with an increase in hematocrit: the yield stress, Newtonian contribution of viscosity, non-Newtonian contribution of viscosity, apparent viscosity and the equilibrium value of the structural parameter which indicates the relative amount of rouleaux in blood. Mathematical equations were developed to give the relationship between parameters and hematocrit. Two other thixotropic parameters, viz. the kinetic rate constant of rouleaux breakdown into individual red cells and the order of the breakdown reaction, were found to be independent of the hematocrit. It is consistent with reaction kinetic theory that the rate constant and the order of reaction are independent of the concentration of reactants.