Microstructure and mechanics of human resistance arteries.

Vascular diseases such as diabetes and hypertension cause changes to the vasculature that can lead to vessel stiffening and the loss of vasoactivity. The microstructural bases of these changes are not presently fully understood. We present a new methodology for stain-free visualization, at a microscopic scale, of the morphology of the main passive components of the walls of unfixed resistance arteries and their response to changes in transmural pressure. Human resistance arteries were dissected from subcutaneous fat biopsies, mounted on a perfusion myograph, and imaged at varying transmural pressures using a multimodal nonlinear microscope. High-resolution three-dimensional images of elastic fibers, collagen, and cell nuclei were constructed. The honeycomb structure of the elastic fibers comprising the internal elastic layer became visible at a transmural pressure of 30 mmHg. The adventitia, comprising wavy collagen fibers punctuated by straight elastic fibers, thinned under pressure as the collagen network straightened and pulled taut. Quantitative measurements of fiber orientation were made as a function of pressure. A multilayer analytical model was used to calculate the stiffness and stress in each layer. The adventitia was calculated to be up to 10 times as stiff as the media and experienced up to 8 times the stress, depending on lumen diameter. This work reveals that pressure-induced reorganization of fibrous proteins gives rise to very high local strain fields and highlights the unique mechanical roles of both fibrous networks. It thereby provides a basis for understanding the micromechanical significance of structural changes that occur with age and disease.

The micromechanics of the superficial zone of articular cartilage.

OBJECTIVE: To investigate the relationships between the unique mechanical and structural properties of the superficial zone of articular cartilage on the microscopic scale. DESIGN: Fresh unstained equine metacarpophalangeal cartilage samples were mounted on tensile and compressive loading rigs on the stage of a multiphoton microscope. Sequential image stacks were acquired under incremental loads together with simultaneous measurements of the applied stress and strain. Second harmonic generation was used to visualise the collagen fibre network, while two photon fluorescence was used to visualise elastin fibres and cells. The changes visualised by each modality were tracked between successive loads. RESULTS: The deformation of the cartilage matrix was heterogeneous on the microscopic length scale. This was evident from local strain maps, which showed shearing between different regions of collagen under tensile strain, corrugations in the articular surface at higher tensile strains and a non-uniform distribution of compressive strain in the axial direction. Chondrocytes elongated and rotated under tensile strain and were compressed in the axial direction under compressive load. The magnitude of deformation varied between cells, indicating differences in either load transmission through the matrix or the mechanical properties of individual cells. Under tensile loading the reorganisation of the elastin network differed from a homogeneous elastic response, indicating that it forms a functional structure. CONCLUSIONS: This study highlights the complexity of superficial zone mechanics and demonstrates that the response of the collagen matrix, elastin fibres and chondrocytes are all heterogeneous on the microscopic scale.

Clostridium perfringensα-toxin interaction with red cells and model membranes.

The effects of Clostridium perfringensα-toxin on host cells have previously been studied extensively but the biophysical processes associated with toxicity are poorly understood. The work reported here shows that the initial interaction between the toxin and lipid membrane leads to measurable changes in the physical properties and morphology of the membrane. A Langmuir monolayer technique was used to assess the response of different lipid species to toxin. Sphingomyelin and unsaturated phosphatidylcholine showed the highest susceptibility to toxin lypolitic action, with a two stage response to the toxin (an initial, rapid hydrolysis stage followed by the insertion and/or reorganisation of material in the monolayer). Fluorescence confocal microscopy on unsaturated phosphatidylcholine vesicles shows that the toxin initially aggregates at discrete sites followed by the formation of localised "droplets" accumulating the hydrolysis products. This process is accompanied by local increases in the membrane dipole potential by about 50 (±42) mV. In contrast, red blood cells incubated with the toxin suffered a decrease of the membrane dipole potential by 50 (±40) mV in areas of high toxin activity (equivalent to a change in electric field strength of 10(7) V m(-1)) which is sufficient to affect the functioning of the cell membrane. Changes in erythrocyte morphology caused by the toxin are presented, and the early stages of interaction between toxin and membrane are characterised using thermal shape fluctuation analysis of red cells which revealed two distinct regimes of membrane-toxin interaction.

The effect of oxidative stress on the membrane dipole potential of human red blood cells.

The membrane dipole potential (ψ(d)) is an important biophysical determinant of membrane function and a sensitive indicator of lipid organisation. In this study we have used the environmentally sensitive probe di-8-anepps to explore the effects of oxidative stress on the membrane dipole potential of human erythrocytes. Cells suspended in 0.15mM phosphate buffered saline containing 0.1mg/ml albumin maintained a mean value for ψ(d) of 270 (±20) mV over the course of 1hour. In the presence of 0.4mM cumene hydroperoxide there was an increase in ψ(d) of 14 (±7)%, accompanied by a decrease in cell diameter of ~14 (±2)%. Exposure of the cells to 0.4mM hydrogen peroxide caused ψ(d) to decrease by 13 (±8)% at the centre of the cell and 8 (±5)% at the edge whilst the diameter remained constant. In both cases the changes were equivalent to a change in transmembrane electric field of a magnitude of ~10MVm(-1), sufficient to influence membrane function. Raman microspectrometry supported the conclusion that cumene exerts its effect primarily on membrane lipids whilst hydrogen peroxide causes the formation of spectrin-haemoglobin complexes which stiffen the membrane.

Motion and mixing for multiple ferromagnetic microswimmers.

This paper concerns the interaction of several ferromagnetic microswimmers, their motion and the resulting fluid mixing. Each swimmer consists of two ferromagnetic beads joined by an elastic link, and is driven by an external, time-dependent magnetic field. The external field provides a torque on a swimmer and, together with the varying attraction between the magnetic beads, generates a time-irreversible motion leading to persistent swimming in a low Reynolds number environment. The aim of the present paper is to consider the interactions between several swimmers. A regime is considered in which identical swimmers move in the same overall direction, and their motion is synchronised because of driving by the external field. It is found that two swimmers tend to encircle one another while three undergo more complicated motion that may involve the braiding of swimmer trajectories. By means of approximations it is established that the interaction between pairs of swimmers gives circulatory motion which falls off with an inverse square law and is linked to their overall speed of motion through the fluid. As groups of two or more swimmers move through the fluid they process fluid, leaving behind a trail of fluid that has undergone mixing: this is investigated by following streak lines numerically.

Biophotonic tools for probing extracellular matrix mechanics.

The complex, hierarchical and heterogeneous biomechanics of the extracellular matrix (ECM) are central to the health of multicellular organisms. Characterising the distribution, dynamics and above all else origins of ECM biomechanics are challenges that have captivated researchers for decades. Recently, a suite of biophotonics techniques have emerged as powerful new tools to investigate ECM biomechanics. In this mini-review, we discuss how the non-destructive, sub-micron resolution imaging capabilities of Raman spectroscopy and nonlinear microscopy are being used to interrogate the biomechanics of thick, living tissues. These high speed, label-free techniques are implemented during mechanical testing, providing unprecedented insight into the compositional and structural response of the ECM to changes in the mechanical environment.

Micro-mechanical damage of needle puncture on bovine annulus fibrosus fibrils studied using polarization-resolved Second Harmonic Generation(P-SHG) microscopy.

Needle injection has been widely used in spinal therapeutic or diagnostic processes, such as discography. The use of needles has been suspected in causing mild disc degeneration which can lead to long-term back pain. However, the localised microscopic damage caused by needles has not been well studied. The local progressive damage on a microscopic level caused by needle punctures on the surface of bovine annulus fibrosus was investigated. Four different sizes of needle were used for the puncture and twenty-nine bovine intervertebral discs were studied. Polarization-resolved second harmonic generation and fluorescent microscopy were used to study the local microscopic structural changes in collagen and cell nuclei due to needle damage. Repeated 70 cyclic loadings at ±5% of axial strain were applied after the needle puncture in order to assess progressive damage caused by the needle. Puncture damage on annulus fibrosus were observed either collagen fibre bundles being pushed aside, being cut through or combination of both with part being lift or pushed in. The progressive damage was found less relevant to the needle size and more progressive damage was only observed using the larger needle. Two distinct populations of collagen, in which one was relatively more organised than the other population, were observed especially after the puncture from skewed distribution of polarization-SHG analysis. Cell shape was found rounder near the puncture site where collagen fibres were damaged.

Feasibility study using surface-enhanced Raman spectroscopy for the quantitative detection of tyrosine and serine phosphorylation.

We investigate the feasibility of colloid-based surface enhanced Raman scattering (SERS) as a highly sensitive technique for detecting peptide phosphorylation at serine and tyrosine residues. Using the recently reported drop-coating deposition Raman method we validate our SERS spectra against normal Raman spectra that would otherwise be unobtainable at such low concentrations. Compared with existing techniques for quantifying peptide phosphorylation, such as high-performance liquid chromatography (HPLC), the short scanning and processing time associated with SERS makes it an attractive alternative for near-real-time measurement at sub micro-molar concentrations. Following pre-processing by Savistky-Golay second derivative (SGSD), the degree of phosphorylation of synthetic peptides is determined using multivariate spectral classification, interval partial least squares (iPLS). Furthermore, our results show that the technique is robust to interference from complex proteins and other phosphorylated compounds present at concentrations typically found in a screening assay.

Spectrin maintains the lateral order in phosphatidylserine monolayers.

We investigate the effect of the skeletal protein spectrin on the lateral order in dipalmitoyl phosphatidylserine monolayers spread on aqueous surfaces using grazing incidence X-ray diffraction. Without spectrin, the condensed lipid monolayer exhibits two-dimensional hexagonal packing, characterized by monotonic decrease in the d-spacing and increase in the degree of order with increasing surface pressure between 17 and 36 mN/m. Addition of spectrin to the aqueous subphase at high pressures preserves the monolayers structural parameters unchanged from 36 to 25 mN/m. These results demonstrate for the first time that spectrin could participate in sustaining the two-dimensional order in lipid domains through a direct interaction with phosphatidylserine species.

The hierarchical response of human corneal collagen to load.

Fibrillar collagen in the human cornea is integral to its function as a transparent lens of precise curvature, and its arrangement is now well-characterised in the literature. While there has been considerable effort to incorporate fibrillar architecture into mechanical models of the cornea, the mechanical response of corneal collagen to small applied loads is not well understood. In this study the fibrillar and molecular response to tensile load was quantified using small and wide angle X-ray scattering (SAXS/WAXS), and digital image correlation (DIC) photography was used to calculate the local strain field that gave rise to the hierarchical changes. A molecular scattering model was used to calculate the tropocollagen tilt relative to the fibril axis and changes associated with applied strain. Changes were measured in the D-period, molecular tilt and the orientation and spacing of the fibrillar and molecular networks. These measurements were summarised into hierarchical deformation mechanisms, which were found to contribute at varying strains. The change in molecular tilt is indicative of a sub-fibrillar "spring-like" deformation mechanism, which was found to account for most of the applied strain under physiological and near-physiological loads. This deformation mechanism may play an important functional role in tissues rich in fibrils of high helical tilt, such as skin and cartilage. STATEMENT OF SIGNIFICANCE: Collagen is the primary mediator of soft tissue biomechanics, and variations in its hierarchical structure convey the varying amounts of structural support necessary for organs to function normally. Here we have examined the structural response of corneal collagen to tensile load using X-rays to probe hierarchies ranging from molecular to fibrillar. We found a previously unreported deformation mechanism whereby molecules, which are helically arranged relative to the axis of their fibril, change in tilt akin to the manner in which a spring stretches. This "spring-like" mechanism accounts for a significant portion of the applied deformation at low strains (<3%). These findings will inform the future design of collagen-based artificial corneas being developed to address world-wide shortages of corneal donor tissue.

Measurement of pulmonary capillary blood flow in infants by plethysmography.

An accurate method for measuring effective pulmonary capillary blood flow (Qc eff) in infants has been developed with an adaptation of the plethysmographic technique. Measurements were made on 19 preterm. 14 small-for-dates, and 7 fullterm normal infants with a constant volume whole body plethysmograph in which the infant rebreathed nitrous oxide. There was a highly significant correlation between Qc eff and body weight, and this relationship was unaffected by premature delivery or intrauterine growth retardation. Mean Qc eff in preterm, small-for dates, and fullterm infants was 203, 208 and 197 ml min-1 kg-1, respectively, with no significant differences between the groups. A significant negative correlation existed between Qc eff and haematocrit in the preterm infants. There was no relationship between weight standardized Qc eff and postnatal age in any of the groups. With this technique, it was possible to readily recognise the presence of rapid recirculation (indicative of shunting) in several of the infants, suggesting that rebreathing methods for the assessment of Qc eff should not be applied indiscriminately during the neonatal period. By taking care to overcome the potential sources of technical error, it was possible to obtain highly reproducible results of Qc eff in infants over a wider age range than has been previously reported.

Viscoelastic properties of insoluble amphiphiles at the air/water interface.

The effects of the presence of a molecular monolayer on the dilatational properties of the air/water interface have been investigated. Two water insoluble amphiphiles, dipalmitoyl phosphatidyl choline and quercetin 3-O-palmitate, were spread onto a pendant drop and the dynamic surface pressure was measured by means of drop shape analysis. The surface dilatational elasticity and viscosity of the spread monolayers were also determined by the oscillating drop technique. Constraints on the range of measuring conditions were investigated and we demonstrated that the pressure-area isotherms derived from oscillatory dynamic measurements display phase behaviour similar to that found in equilibrium measurements, albeit at reduced resolution. Both the amphiphiles formed purely elastic films that were characterised by a dilatational modulus that depended on the surface concentration and obeyed a power scaling law. The exponent of the relationship could be related to the thermodynamic conditions prevailing at the interface. The phospholipid monolayer scaling exponent was 2.8 in a temperature range of 20-26 degrees C indicates a favourable solvency of molecules in the bidimensional matrix. A very high scaling exponent (11.8 at 7 degrees C) for quercetin palmitate was interpreted assuming that molecules self-organise in fibre-like structures. This interface structure and the phase behaviour was found consistent with observations of the surface film obtained by Brewster angle microscopy. The structured quercetin 3-O-palmitate monolayers are disrupted by temperature increase or by adding a 0.2 molar fraction of the immiscible dipalmitoyl phosphatidyl choline.

Reversible and irreversible interactions between elastin and plasma lipoproteins.

The interactions between radiolabeled, human plasma lipoproteins and elastin derived from bovine ligamentum nuchae were investigated using a washout technique. The interaction was characterised by Ki, a coefficient of irreversible binding, and Kr, the reversible partition coefficient. For both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) the Ki values decreased as total lipoprotein concentration increased, suggesting that the binding is saturable, and were similar in magnitude to those measured by other workers using elastin derived from the human aorta. For both LDL and HDL the Kr values were independent of lipoprotein concentration in the range 0.1 microgram/ml-1.5 micrograms/ml. At a total protein concentration of 1.5 mg/ml in the incubation medium, the reversible interactions were comparable in magnitude to the irreversible.

High resolution imaging of the distribution and permeability of methyl viologen dication in bovine articular cartilage using scanning electrochemical microscopy.

Scanning electrochemical microscopy (SECM) has been used in the induced transfer (SECMIT) mode to image the permeability of a probe cation, methyl viologen (MV(2+)), in samples of articular cartilage. An ultramicroelectrode (UME), scanned just above the surface of a sample, is used to amperometrically detect the probe solute. The resulting depletion of MV(2+) in solution induces the transfer of this cation from the sample into the solution for detection at the UME. The current provides quantitative information on local permeability, provided that the sample-UME distance is known. It is shown that the necessary topographical information can be obtained using the amperometric response for the oxidation of Ru(CN)(4-)(6), which does not permeate into the cartilage matrix. This procedure was validated by marking samples in situ, after electrochemical imaging, with subsequent examination by ex situ interferometry and optical microscopy. Wide variations in the permeability of MV(2+) have been detected by SECMIT. These observations represent the first demonstration of the inhomogeneous permeability of a cation in cartilage on a micrometre scale. The permeability maps show similar features to the proteoglycan distribution, identified by toluidine blue staining, and it is likely that proteoglycans are the main determinant of MV(2+) permeability in articular cartilage.

Transport of radioactive oxygen, nitrogen and xenon into the rabbit thoracic aorta in situ.

An in situ preparation of the rabbit thoracic aorta has been used to study the transport of short-lived radioactive isotopes of oxygen (15O16O), nitrogen (13N14N) and an isotope of xenon (133Xe) from 0.9% NaCl into the artery wall. At a luminal hydrostatic pressure of 2 kPa the uptake of all three isotopes by the intima-media increased with time in experiments of up to 15 min duration. Uptake of labelled oxygen was an order of magnitude higher than that of xenon and almost two orders of magnitude higher than nitrogen uptake. Adventitial uptake was similar for all three species. For a perfusion time of 2 1/2 min, the intimal-medial uptake of all three gases increased by almost an order of magnitude as luminal pressure increased from 2 to 13 kPa. Adventitial uptake increased rapidly and at a similar rate for all three gases. The studies were limited in depth and accuracy by the small quantities of isotope available.

The use of radioiodinated human serum albumin in measurements of arterial permeability.

The interactions of radioiodinated human serum albumin and sodium iodide with plasma proteins and their uptake by the arterial wall are investigated in vivo and in vitro. The results clearly show that in most situations iodinated albumin is unsuitable for studies on arterial permeability.

Myocardial tissue perfusion determined by particulate and diffusible tracers during ischaemia: what is measured?

OBJECTIVE: Myocardial perfusion can be determined by many techniques which can be broadly divided into those employing particulate tracers and those employing diffusible tracers. The most commonly used particulate tracer is radioactive microspheres. However, as with other particulate tracers, they only determine convective transport from pre-capillary arterioles. If convective transport is the limiting factor in solute exchange, then particulate tracers will give comparable measurements to diffusible tracer techniques. However, if solute transport becomes diffusion-limited or alternative pathways of convective transport become more important, which may occur during regional ischaemia, perfusion visualised with clearance techniques using diffusible tracers may be greater than that determined with particulate tracers. This study set out to investigate this possibility in the rabbit myocardium under normal and ischaemic conditions. METHODS: A pentobarbitone-anaesthetised rabbit model of regional ischaemia was used. Ischaemia of the apical region was induced by ligation of the large left ventricular branch of the circumflex artery. Tissue perfusion was determined by radioactive microspheres (n = 5) and the clearance of hydrogen, which was detected voltammetrically by platinum microelectrodes (n = 5). Measurements were made prior to and following coronary ligation and the ischaemic region was demarcated using the particulate tracer monastral blue. The exchange of diffusible solutes was visualised using digital fluorescence microscopy on histological sections of tissue following systemic administration of the fluorophore Evans blue labelled albumin (n = 4). RESULTS: Coronary ligation produced an ischaemic zone occupying 50 +/- 13% of the left ventricle. In ischaemic tissue, flow determined by microspheres fell to 3.9 +/- 4.1% of its pre-ligation value, but solute exchange fell only to 22 +/- 10% (adjusted for changes in the partition coefficient of H2 during ischaemia, P < 0.05). Perfusion measured by microspheres and hydrogen clearance was unchanged in the non-ischaemic area during coronary ligation. There was preferential uptake of Evans blue albumin towards the endocardial surface in the ischaemic region and areas of local uptake through the ventricular wall, which were possibly associated with vessels. CONCLUSION: This work demonstrates that under normal physiological conditions nutrient supply is determined by pre-capillary delivery. However, during ischaemia diffusive transport plays an increasingly important role. The alternative pathways for solute exchange are likely to have an important influence on the rate and extent of myocardial necrosis during coronary occlusion.

Effects of particle size and perfusate composition on the uptake of colloidal gold by the rabbit thoracic aorta perfused in situ.

The influence has been investigated of particle size on the uptake of radioactive gold colloid by the rabbit thoracic aorta perfused in situ. Particles ranging in diameter from 14 nm to 40 nm were suspended in 0.9% NaCl and infused either at a pressure of 15 mm Hg for times of between 2 1/2 and 60 min or at pressure of between 15 and 160 mm Hg for 5 min. Uptake by the whole intima-media increased with perfusion time and hydrostatic pressure but did not depend on particle size. Radioactive assay of serial sections across the aortic wall also showed that particle size did not influence the distribution of tracer. An effect of perfusate composition on uptake was demonstrated in further experiments in which particles either 14 or 40 nm in diameter were suspended in pooled rabbit serum and infused at pressures of between 15 and 140 mm Hg for 5 min. Uptake and transmural distribution were again independent of particle size, but uptake was 4-5-fold less than when the particles were perfused in saline. Under all perfusion conditions radioactivity fell steeply across the intima and then rose gradually across the media and adventitia. Radioactivity in the outer media and adventitia increased with perfusion time but little change could be detected in intimal activity. In transmission electron micrographs, particles in the intima were not seen to penetrate the internal elastic lamella and in the outer media particles remained extracellular and did not enter collagen bundles. Autoradiographs showed that particles in the intima were uniformly distributed around the circumference of the vessel but in the outer media and adventitia particles usually clustered close to the vasa vasorum.

Net albumin transport across the wall of the rabbit common carotid artery perfused in situ.

We have studied the transport of radioactively labelled albumin in the rabbit common carotid artery perfused in situ at 100 cm H2O luminal pressure in the anaesthetized living animal, assessing the distribution of concentration across the wall by means of sequential frozen sectioning. We have compared the findings with those of experiments in which we have attempted to saturate the wall with label. Our findings support the belief that there is a net transport of macromolecules across the arterial wall. They show in addition that the wall is inhomogeneous. The distribution volume for label is greater in the adventitia than the media, which appears to offer a larger resistance. The transport process is seemingly dominantly diffusional.

Effects of perfusate composition on binding of ruthenium red and gold colloid to glycocalyx of rabbit aortic endothelium.

The effects of perfusate composition on some of the properties of the endothelial glycocalyx were investigated electron microscopically. Rabbit aorta was perfused in situ in preparation for fixation with 1% OsO4 containing 0.1 Ruthenium red (RR) or perfusion with wheat germ agglutinate (WGA)-coated gold particles suspended in 0.9% NaCl, Tyrode's solution, or pooled rabbit serum. Intense RR staining of the intimal glycocalyx was found only in vessels that had been exposed to 150 mmol/L NaCl, pH 7.4, or to albumin (4.0 g/100 ml), gamma-globulin (0.7 g/100 ml), or fibrinogen (0.3 g/100 ml) in Tyrode's solution, pH 7.4, for 5 min prior to fixation. The adherence of WGA-coated gold particles to the intimal and vasa vasorum glycocalyces was found in saline- and Tyrode's-perfused vessels. The density of the WGA-coated gold particles was greatest in the glycocalyx of damaged endothelium. In vessels flushed with serum, no RR stain or WGA-coated gold colloid was seen in the glycocalyx. This study demonstrates that saline perfusion facilitates the passage of RR and WGA-coated gold particles into the endothelial glycocalyx. We conclude that serum proteins and cell-surface glycoprotein chains act cooperatively to impede the access of some molecules to the endothelial surface.