High-resolution cell outline segmentation and tracking from phase-contrast microscopy images.

Accurate extraction of cell outlines from microscopy images is essential for analysing the dynamics of migrating cells. Phase-contrast microscopy is one of the most common and convenient imaging modalities for observing cell motility because it does not require exogenous labelling and uses only moderate light levels with generally negligible phototoxicity effects. Automatic extraction and tracking of high-resolution cell outlines from phase-contrast images, however, is difficult due to complex and non-uniform edge intensity. We present a novel image-processing method based on refined level-set segmentation for accurate extraction of cell outlines from high-resolution phase-contrast images. The algorithm is validated on synthetic images of defined noise levels and applied to real image sequences of polarizing and persistently migrating keratocyte cells. We demonstrate that the algorithm is able to reliably reveal fine features in the cell edge dynamics.

Stretch-elicited calcium responses in the intact mouse thoracic aorta.

Stretch-elicited intracellular calcium ([Ca(2+)](i)) changes in individual smooth muscle cells in a ring of aorta were measured simultaneously with the force developed by the ring. A phasic increase in [Ca(2+)](i) was observed in 30% of the cells and a sustained one in 10%. Depletion of intracellular calcium store by thapsigargin and caffeine decreased phasic and increased sustained calcium responses. The inhibition of calcium entry either by stretching the aorta in a calcium-free medium or by the inhibition of stretch-activated, non-selective cationic channels by 5 microM GsMtx-4 toxin, decreased the proportion of sustained [Ca(2+)](i) responses but increased transient responses. In this condition, a third of the cells responded to stretch by a bursts of [Ca(2+)](i) spikes. The decrease of calcium influx triggered the generation of burst of calcium spikes after the application of stretch steps to the vascular wall. We conclude that progressive recruitment of smooth muscle cells is the mechanism underlying the force-generating part of the myogenic response. Two types of stretch-elicited calcium responses were observed during the recruitment of the smooth muscle cells. One was a phasic calcium discharge generated by the sarcoplasmic reticulum. The second was a tonic response produced by the activation of the stretch-sensitive cationic channels allowing extracellular Ca(2+) entry.

Myofibroblast development is characterized by specific cell-cell adherens junctions.

Myofibroblasts of wound granulation tissue, in contrast to dermal fibroblasts, join stress fibers at sites of cadherin-type intercellular adherens junctions (AJs). However, the function of myofibroblast AJs, their molecular composition, and the mechanisms of their formation are largely unknown. We demonstrate that fibroblasts change cadherin expression from N-cadherin in early wounds to OB-cadherin in contractile wounds, populated with alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts. A similar shift occurs during myofibroblast differentiation in culture and seems to be responsible for the homotypic segregation of alpha-SMA-positive and -negative fibroblasts in suspension. AJs of plated myofibroblasts are reinforced by alpha-SMA-mediated contractile activity, resulting in high mechanical resistance as demonstrated by subjecting cell pairs to hydrodynamic forces in a flow chamber. A peptide that inhibits alpha-SMA-mediated contractile force causes the reorganization of large stripe-like AJs to belt-like contacts as shown for enhanced green fluorescent protein-alpha-catenin-transfected cells and is associated with a reduced mechanical resistance. Anti-OB-cadherin but not anti-N-cadherin peptides reduce the contraction of myofibroblast-populated collagen gels, suggesting that AJs are instrumental for myofibroblast contractile activity.

Intercellular ultrafast Ca(2+) wave in vascular smooth muscle cells: numerical and experimental study.

Vascular smooth muscle cells exhibit intercellular Ca(2+) waves in response to local mechanical or KCl stimulation. Recently, a new type of intercellular Ca(2+) wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca(2+) wave and it was suggested to be the result of the interplay between membrane potential and Ca(2+) dynamics which depended on influx of extracellular Ca(2+), cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca(2+) wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca(2+) wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca(2+) wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca(2+) waves in smooth muscle cells.

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells.

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.

Cytosolic-free calcium in smooth-muscle and endothelial cells in an intact arterial wall from rat mesenteric artery in vitro.

The regulation of cytosolic-free calcium concentration of smooth-muscle and endothelial cells was mainly studied on cultured cells where the cross talk between these two coupled cell types is lost. In the present study, the cytosolic-free calcium concentration in the endothelial and the smooth-muscle cells was examined in an intact arterial wall in vitro. Strips of the main branch of rat mesenteric artery were used. Cytosolic-free calcium concentration [Ca2+]i was estimated by determining the fluorescence ratio of the two calcium probes, Fluo-4 and Fura red. The emitted fluorescence of both probes was measured with a confocal microscope. We showed that potassium and phenylephrine, which increase the cytosolic -free calcium concentration of the smooth-muscle cells, also indirectly influence the calcium concentration in the endothelial cells. By simultaneously determining [Ca2+]i in the endothelial and the smooth-muscle cells of an arterial strip, we observed that when calcium increases in the endothelial cells in response to acetylcholine, it slightly decreases in the smooth-muscle cells. We conclude that the regulation of [Ca2+]i in the arterial endothelial cell, depends according to the stimuli either upon the endothelial cells themselves, or upon the coupled smooth-muscle cells.

Evaluation of arterial compliance-pressure curves. Effect of antihypertensive drugs.

A new high-precision ultrasonic device was developed to determine noninvasively arterial compliance as a function of blood pressure. Because of the nonlinear elastic properties of arterial walls, measurements of compliance can be appropriately compared only if obtained over a range of pressures. This apparatus was used to evaluate in a double-blind, parallel fashion the effect of three different antihypertensive drugs and of a placebo on radial artery compliance. Thirty-two normotensive volunteers were randomly allocated to an 8-day, once-a-day oral treatment with either a placebo, 100 mg atenolol, 20 mg nitrendipine, or 20 mg lisinopril. Blood pressure, heart rate, radial artery diameter, and arterial compliance were measured immediately before as well as 6 hours after dosing on the first and last days of the study. On the eighth day of administration, within 6 hours after dosing, lisinopril induced an acute increase in radial artery diameter, from 2.99 +/- 0.06 to 3.28 +/- 0.09 mm (mean +/- SEM, p less than 0.01). The compliance-pressure curve was shifted upward on day 1 (p less than 0.01) as well as on day 8 (p less than 0.05). None of the other drugs induced any significant modification of these parameters. Arterial compliance has a strong nonlinear dependency on intra-arterial pressure and therefore has to be defined as a function of pressure. Antihypertensive drugs acting by different mechanisms may have different effects on the mechanical properties of large arteries.

Theoretical study of dynamics of arterial wall remodeling in response to changes in blood pressure.

The dynamics of arterial wall remodeling was studied on the basis of a phenomenological mathematical model. Sustained hypertension was simulated by a step increase in blood pressure. Remodeling rate equations were postulated for the evolution of the geometrical dimensions that characterize the zero stress state of the artery. The driving stimuli are the deviations of the extreme values of the circumferential stretch ratios and the average stress from their values at the normotensive state. Arterial wall was considered to be a thick-walled tube made of nonlinear elastic incompressible material. Results showed that thickness increases montonically with time whereas the opening angle exhibits a biphasic pattern. Geometric characteristics reach asymptotically a new homeostatic steady state, in which the stress and strain distribution is practically identical with the distribution under normotensive conditions. The model predictions are in good agreement with published experimental findings.

Static compression of articular cartilage can reduce solute diffusivity and partitioning: implications for the chondrocyte biological response.

Chondrocytes depend upon solute transport within the avascular extracellular matrix of adult articular cartilage for many of their biological activities. Alterations to bioactive solute transport may, therefore, represent a mechanism by which cartilage compression is transduced into cellular metabolic responses. We investigated the effects of cartilage static compression on diffusivity and partitioning of a range of model solutes including dextrans of molecular weights 3 and 40 kDa, and tetramethylrhodamine (a 430 Da fluorophore). New fluorescence methods were developed for real-time visualization and measurement of transport within compressed cartilage explants. Experimental design allowed for multiple measurements on individual explants at different compression levels in order to minimize confounding influences of compositional variations. Results demonstrate that physiological levels of static compression may significantly decrease solute diffusivity and partitioning in cartilage. Effects of compression were most dramatic for the relatively high molecular weight solutes. For 40 kDa dextran, diffusivity decreased significantly (p<0.01) between 8% and 23% compression, while partitioning of 3 and 40 kDa dextran decreased significantly (p<0.01) between free-swelling conditions and 8% compression. Since diffusivity and partitioning can influence pericellular concentrations of bioactive solutes, these observations support a role for perturbations to solute transport in mediating the cartilage biological response to compression.

Nonlinear separation of forward and backward running waves in elastic conduits.

A new method for the separation of forward and backward running waves in elastic conduits, with possible extension to the arterial system, has been developed. The mathematical model is based on the one-dimensional flow equations which allow the treatment of non-periodic or transient pressure and flow pulses. The method is fully nonlinear, i.e. no linearizing assumptions are made. The method includes the effects of convective acceleration and pressure-dependent vessel compliance. A first approximation for the fluid friction at the wall is also included. The application of the method requires the knowledge of the elastic properties, the instantaneous pressure and flow, as well as the instantaneous spatial derivatives of pressure and flow. Analysis of simulated data shows good results and suggests that the proposed method, unlike previous quasi-nonlinear and frequency domain methods, can be applied to strongly nonlinear and/or nonperiodic flows. The method predicts that if a linear analysis is applied to a nonlinear system errors arise.

Modeling of the wave transmission properties of large arteries using nonlinear elastic tubes.

We propose a new, simple way of constructing elastic tubes which can be used to model the nonlinear elastic properties of large arteries. The tube models are constructed from a silicon elastomer (Sylgard 184, Dow Corning), which exhibits a nonlinear behavior with increased stiffness at high strains. Tests conducted on different tube models showed that, with the proper choice of geometric parameters, the elastic properties, in terms of area-pressure relation and compliance, can be similar to that of real arteries.

Residual strain effects on the stress field in a thick wall finite element model of the human carotid bifurcation.

A three-dimensional finite element model of the carotid artery bifurcation was constructed in order to determine the stress field and assess the modification of the stress field when residual strain is taken into account. Residual strain in the carotid bifurcation was characterized by experimental observations. According to these observations, a geometrical model of the carotid artery was constructed to exhibit a state free of strain. Appropriate boundary conditions were applied to yield the correct geometry in the unloaded state, and physiological levels of pressure and axial stretching were applied. The model took into account the varying thickness of the arterial wall along the bifurcation. For modeling purposes, the material was considered to be hyperelastic, incompressible, homogenous and isotropic. For comparison, a similar model of the carotid artery which does not include the effects of residual strain was also created. The results demonstrate that in the model of the carotid artery bifurcation with residual strain, the distribution of maximum principal stress along the inner wall and the circumferential stress throughout the wall is much more uniform than in the model without residual strain. The ratio between the stress at the inner and the outer walls is highest at the lateral wall of the carotid sinus; this is the same location known to be a site of low and oscillatory fluid wall shear stress, and the principal location of early intimal thickening. These results suggest that the localization of atherosclerosis in the carotid artery may be due to local variations in both fluid wall shear stress and solid wall stress.

The effects of time-varying curvature on velocity profiles in a model of the coronary arteries.

Blood flow patterns in the coronary arteries are of interest due to their possible involvement in atherosclerosis localization. Spatial variations in hemodynamic flow patterns are predominantly determined by the vessel geometry. The coronary arteries represent a unique situation in the cardiovascular system because their geometry undergoes large dynamic variations during each cardiac cycle due to the contraction of the heart. This study was initiated to analyze the effects of time-varying curvature on flow velocity profiles in a curved tube model of the coronary arteries. An in vitro flow model was constructed, which consisted of a flexible curved tube through which fluid flowed under a steady imposed pressure gradient. The radius of curvature of the tube was varied in time using a stepper motor and carriage. Two different deformation configurations were used to determine if variations in center of curvature displacement affected the velocity profiles. It was found in both cases that the skewing of the axial velocity profile depended on the instantaneous dynamic vessel movement, with maximal skewing occurring when the radius of curvature was in transition from the minimum to the maximum value. The change in skewing was greater for the case where carriage moved obliquely to the main direction of flow than when the carriage moved perpendicularly. Although this study was limited to relatively low values of the radius of curvature and change in curvature, an initial understanding of this flow situation was obtained which may lead to the development of more physiologic models.

Effects of friction and nonlinearities on the separation of arterial waves into their forward and backward components.

In this paper we examine the importance of fluid friction and nonlinearities due to the area-pressure relationship and to the convective acceleration on the separation of arterial pressure and flow waves into their forward and backward components. Experiments were run in straight uniform nonlinearly elastic tubes. Different degrees of fluid friction and nonlinearities, covering the physiological range, have been tested. We predicted the forward and backward running pressure components using two wave separation methods: the classical linear method (Westerhof et al., Cardiovasc. Res, 6,648-656, 1972) and the first order correction (FOC) method (Pythoud et al., Trans ASME J. Biomech. Engng, in press) which takes nonlinearities and fluid friction into account. We found that the two methods yield somewhat different predictions. The differences tend to increase with the degree of fluid friction and nonlinearities and are typically of the order of 4-8%. We further compared the transmission ratio of forward and backward waves predicted by both methods. The transmission ratio was found to be overestimated by 10% by the classical linear method. The nonlinear method gave more accurate estimates, consistent with theory. We conclude that, for in vivo applications, the classical linear method should be the method of choice because it is simpler to use and the erros involved (4-8%) are comparable to measurement erros.

On the wave transmission and reflection properties of stenoses.

This study is concerned with the wave reflection properties of arterial stenoses. Two theoretical models have been developed for deriving the reflection coefficient: a linear model resulting from the linearization of the pressure drop-flow equation and an indirect, quasi-nonlinear model, based on the separation of pressure waves into their forward and backward running components proximal and distal to the stenosis. The linear method gave consistently lower values for the reflection coefficient when compared to the quasi-nonlinear model. In vitro experiments in elastic tubes showed that the reflection coefficient is strongly dependent on stenosis severity, mean flowrate, and the elastic properties of the proximal unobstructed artery. For critical stenoses the reflection coefficient is frequency and pulsatility independent. The results suggest that hemodynamically nonsevere stenoses may cause significant wave reflections.

In vitro diameter response of rat femoral artery to flow in the presence and absence of endothelium.

We have examined the diameter response of rat femoral artery segments in the presence and absence of endothelium to changes in flow rate. The segments were isolated, mounted on microcannulae, maintained at 37 degrees C, and perfused at 90 mmHg with Tyrode's solution. The external arterial diameter was measured using video-microscopy. The mean control diameter was 741+/-22 microm (mean+/-SEM,n=7). The arteries were preconstricted to 75+/-1% of the control diameter with a superfusion of 1 microM norepinephrine (NE). Endothelial function was verified by perfusion of 1 micro;M acetylcholine (ACh). Two different flow protocols were employed: step changes in flow (n=7) and low-frequency sinusoidal flow changes (0.01Hz0.05). Sinusoidal flow oscillations resulted in sinusoidal diameter oscillations, whose amplitude and phase lag were inversely proportional to the frequency of the flow oscillations. A first-order low-pass filter, with a time constant of 28+/-3 and 30+/-5s for arteries with and without functional endothelium, respectively, was used to describe the relation between oscillatory flow and diameter. The response of the rat femoral arteries to changes in flow was not found to be different whether the endothelium was intact or removed.

Velocity vector reconstruction for color flow Doppler: experimental evaluation of a new geometrical method.

Ultrasonic color flow mapping (CFM) is employed for the diagnosis of a variety of cardiovascular abnormalities. But CFM permits only the analysis of the in-beam component of the velocity distribution. A reconstruction method based on a geometrical approach was developed to calculate the two-dimensional, in-plane velocity vector using the information of two independent CFM measurements obtained from two closely spaced transducers. However, with the CFM measurements being limited by the noise and quantified in a limited number of velocity values, the accuracy of the velocity vector reconstruction is not satisfactory. A simple filtering process was applied to improve the results. The method was tested with simulations and a series of in vitro measurements. The results of the simulations were compared with analytical solutions. The in vitro measurements were compared with the results of laser Doppler anemometry (LDA) and theoretical calculations. The results of in vivo measurements were compared with theoretical calculations based on Womersley's theory. The comparison showed good applicability of the method to different flow fields. The flow vector reconstructed without filtering had an error of more than 20%; whereas our method, including the filtering process, had an error of less than 10%, in comparison with the theoretical or the LDA results. The method could be extended to three-dimensional flows.

Microplates: a new tool for manipulation and mechanical perturbation of individual cells.

We present a new type of microinstrument allowing manipulation and mechanical perturbation of individual cells under an optical microscope. These instruments, which we call microplates, are pulled from rectangular glass bars. They have flat tips, typically 2 microm thick x 20 microm wide, whose specific shape and stiffness can be adjusted through the pulling protocol. After appropriate chemical treatment, microplates can support cell adhesion and/or spreading. Rigid microplates are used to hold cells, whereas more flexible ones serve as stress sensors, i.e. their deflexion is used to probe forces in the range of 1-1000 nN. The main advantages of microplates are their simple geometry and surface properties, and their ability to provide mechanical measurements. In this methodological paper, we give details about microplate preparation and adhesiveness, manipulation set-up, force calibration, and image analysis. Several manipulations have already been carried out on fibroblasts, including uniaxial deformation, micropipet aspiration of adherent cells, and cell-substrate separation. Our results to date provide new insights into the morphology, mechanical properties, and adhesive resistance of cells. Many future applications can be envisaged.

Comparison between segmental and selective blood flow volume of the lower limbs: a plethysmographic and ultrasonic study of normal subjects at rest.

In order to find the correlations existing between segmental and selective arterial blood flow volume, 20 healthy subjects have been submitted to calf venous occlusion plethysmography and Multichannel Digital Pulsed Doppler examination of the common femoral and popliteal arteries at rest. We found a linear correlation only between mean popliteal artery blood flow volume and plethysmographic calf blood flow volume (r = 0,41; p less than 0,01). We therefore suggest that venous occlusion plethysmography should not be used for the assessment of aorto-iliac and common femoral hemodynamics after direct arterial reconstructive surgery. Its use should be complementary to pulsed Doppler blood flow volume determination at the femoro-popliteal level, along with the usual pressure measurement.

Pulsed Doppler assessment of deep femoral artery hemodynamics: a preliminary report.

The functional capability of the deep femoral artery (DFA) as a collateral channel is an important feature in patients suffering peripheral arterial occlusive disease (PAOD). A noninvasive method of indirect quantification of the DFA blood flow volume is presented. In normal lower limbs, it was found that the common femoral artery (CFA) mean blood flow volume was maintained at 59% of its resting value when the superficial femoral artery (SFA) was occluded by a pneumatic cuff placed around the thigh. The reduction of the mean flow volume is mainly due to an increase of the diastolic backward flow, while the maximum systolic flow remains constant. In a few patients with various degrees of stenosis of the DFA, we observed a much greater drop in mean CFA blood flow volume than in normal subjects. On the basis of this preliminary study, we conclude that: --When the normal SFA is occluded, pulsatile flow through the CFA is maintained. --Occlusion flow reflects the runoff and the compliance of the DFA bed. --Occlusion flow is diminished in patients with DFA stenosis. It is suggested that this non-invasive test might help the physician to assess the involvement of the DFA in PAOD. Further investigation is needed to establish the correlation between quantitative occlusion blood flow volume and the degree of DFA stenosis.