Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer.

Cyclic stretch is known to alter a number of cellular and subcellular processes, including those involved in nonviral gene delivery. We have previously shown that moderate equibiaxial cyclic stretch (10% change in basement membrane area, 0.5 Hz, 50% duty cycle) of human pulmonary A549 cells enhances gene transfer and expression of reporter plasmid DNA in vitro, and that this phenomena may be due to alterations in cytoplasmic trafficking. Although the path by which plasmid DNA travels through the cytoplasm toward the nucleus is not well understood, the cytoskeleton and the constituents of the cytoplasm are known to significantly hinder macromolecular diffusion. Using biochemical techniques and immunofluorescence microscopy, we show that both the microfilament and microtubule networks are significantly reorganized by equibiaxial cyclic stretch. Prevention of this reorganization through the use of cytoskeletal stabilizing compounds mitigates the stretch-induced increase in gene expression, however, depolymerization in the absence of stretch is not sufficient to increase gene expression. These results suggest that cytoskeletal reorganization plays an important role in stretch-induced gene transfer and expression.

Steady-state pleural fluid flow and pressure and the effects of lung buoyancy.

Both theoretical and experimental studies of pleural fluid dynamics and lung buoyancy during steady-state, apneic conditions are presented. The theory shows that steady-state, top-to-bottom pleural-liquid flow creates a pressure distribution that opposes lung buoyancy. These two forces may balance, permitting dynamic lung floating, but when they do not, pleural-pleural contact is required. The animal experiments examine pleural-liquid pressure distributions in response to simulated reduced gravity, achieved by lung inflation with perfluorocarbon liquid as compared to air. The resulting decrease in lung buoyancy modifies the force balance in the pleural fluid, which is reflected in its vertical pressure gradient. The data and model show that the decrease in buoyancy with perfluorocarbon inflation causes the vertical pressure gradient to approach hydrostatic. In the microgravity analogue, the pleural pressures would be toward a more uniform distribution, consistent with ventilation studies during space flight. The pleural liquid turnover predicted by the model is computed and found to be comparable to experimental values from the literature. The model provides the flow field, which can be used to develop a full transport theory for molecular and cellular constituents that are found in pleural fluid.

A system to impose prescribed homogenous strains on cultured cells.

There is presently significant interest in cellular responses to physical forces, and numerous devices have been developed to apply stretch to cultured cells. Many of the early devices were limited by the heterogeneity of deformation of cells in different locations and by the high degree of anisotropy at a particular location. We have therefore developed a system to impose cyclic, large-strain, homogeneous stretch on a multiwell surface-treated silicone elastomer substrate plated with pulmonary epithelial cells. The pneumatically driven mechanism consists of four plates each with a clamp to fix one edge of the cruciform elastomer substrate. Four linear bearings set at predetermined angles between the plates ensure a constant ratio of principal strains throughout the stretch cycle. We present the design of the device and membrane shape, the surface modifications of the membrane to promote cell adhesion, predicted and experimental measurements of the strain field, and new data using cultured airway epithelial cells. We present for the first time the relationship between the magnitude of cyclic mechanical strain and the extent of wound closure and cell spreading.

5-Hydroxytryptamine-induced microvascular pressure transients in lungs of anaesthetized rabbits.

We determined lung microvascular pressure transients induced by 5-hydroxytryptamine (5HT), by the micropuncture technique. We mechanically ventilated anaesthetized (halothane 0.8%), open-chested rabbits, in which we recorded pulmonary artery (PA), left atrial (LA) and carotid artery pressures and cardiac output. For 4-min periods of stopped ventilation, we constantly inflated the lung with airway pressure of 7 cmH2O, then micropunctured the lung to determine pressures in arterioles and venules of 20-25 microm diameter. An intravenous bolus infusion of 5HT (100 microg), increased total pulmonary vascular resistance by 59%. Prior to 5HT infusion, the arterial, microvascular and venous segments comprised 30, 50 and 19% of the total pulmonary vascular pressure drop, respectively. However 14 s after 5HT infusion, the PA-arteriole pressure difference (arterial pressure drop) increased 46%, while the venule-LA pressure difference (venous pressure drop) increased >100%. The arteriole-venule pressure difference (microvascular pressure drop) was abolished. The increase in the arterial pressure drop was maintained for 4.8 min, whereas the increased venous pressure drop reverted to baseline in <1 min. We conclude that in the rabbit lung in situ, a 5HT bolus causes sustained arterial constriction and a strong but transient venous constriction.

A rat lung model of instilled liquid transport in the pulmonary airways.

When a liquid is instilled in the pulmonary airways during medical therapy, the method of instillation affects the liquid distribution throughout the lung. To investigate the fluid transport dynamics, exogenous surfactant (Survanta) mixed with a radiopaque tracer is instilled into tracheae of vertical, excised rat lungs (ventilation 40 breaths/min, 4 ml tidal volume). Two methods are compared: For case A, the liquid drains by gravity into the upper airways followed by inspiration; for case B, the liquid initially forms a plug in the trachea, followed by inspiration. Experiments are continuously recorded using a microfocal X-ray source and an image-intensifier, charge-coupled device image train. Video images recorded at 30 images/s are digitized and analyzed. Transport dynamics during the first few breaths are quantified statistically and follow trends for liquid plug propagation theory. A plug of liquid driven by forced air can reach alveolar regions within the first few breaths. Homogeneity of distribution measured at end inspiration for several breaths demonstrates that case B is twice as homogeneous as case A. The formation of a liquid plug in the trachea, before inspiration, is important in creating a more uniform liquid distribution throughout the lungs.

Partial prevention of monocyte and granulocyte activation in experimental vein grafts by using a biomechanical engineering approach.

Leukocytes interact with endothelial cells and contribute to the development of vascular diseases such as thrombosis and atherosclerosis. These processes are possibly influenced by mechanical factors. This study focused on the role of mechanical stretch in the activation of monocytes and granulocytes in experimental vein grafts. Two models were created by using rats: a nonengineered vein graft with increased tensile stress, which was created by grafting a jugular vein into the abdominal aorta, and an engineered vein graft with reduced tensile stress, which was created by restricting the vein graft into a cylindrical sheath constructed by using fixative-treated intestinal tissue. The density of activated monocytes and granulocytes, which attached to the endothelium, and the distribution of the intercellular adhesion molecule (ICAM)-1 in endothelial cells were examined using immunohistological assays. It was found that, in nonengineered vein grafts, the density of activated monocytes and granulocytes increased significantly compared to that in normal jugular veins at day 1, 5, 10 and 20. At each observation time, the cell density in the proximal region of the nonengineered vein grafts was significantly higher than that in the middle and distal regions, and the cell density in the distal region was significantly higher than that in the middle region. These changes were associated with ICAM-1 clustering at day 1 and 5 and focal ICAM-1 un-regulation at day 10 and 20. In engineered vein grafts, the density of activated monocytes and granulocytes decreased significantly compared to that in nonengineered vein grafts at all observation times, although it was significantly higher than that in normal jugular veins. At each observation time, the cell density in the proximal and distal regions was significantly higher than that in the middle region, but no significant difference was found between the proximal and distal regions. ICAM-1 clustering along endothelial cell borders was found at day 1 and 5, but no apparent focal ICAM-1 up-regulation was found at day 10 and 20. These results suggested that mechanical stretch due to exposure to increased tensile stress contributed to the activation of monocytes and granulocytes in experimental vein grafts, and this event could be partially prevented by reducing tensile stress using a biomechanical engineering approach.

Surfactant-spreading and surface-compression disturbance on a thin viscous film.

Spreading of a new surfactant in the presence of a pre-existing surfactant distribution is investigated both experimentally and theoretically for a thin viscous substrate. The experiments are designed to provide a better understanding of the fundamental interfacial and fluid dynamics for spreading of surfactants instilled into the lung. Quantitative measurements of spreading rates were conducted using a fluorescent new surfactant that was excited by argon laser light as it spread on an air-glycerin interface in a petri dish. It is found that pre-existing surfactant impedes surfactant spreading. However, fluorescent microspheres used as surface markers show that pre-existing surfactant facilitates the propagation of a surface-compression disturbance, which travels faster than the leading edge of the new surfactant. The experimental results compare well with the theory developed using lubrication approximations. An effective diffusivity of the thin film system is found to be Deff = (E*gamma)/(mu/H), which indicates that the surface-compression disturbance propagates faster for larger background surfactant concentration, gamma, larger constant slope of the sigma*-gamma* relation, -E*, and smaller viscous resistance, mu/H. Note that sigma* and gamma* are the dimensional surface tension and concentration, respectively, mu is fluid viscosity, and H is the unperturbed film thickness.

Influence of intravenous perfluorocarbon administration on the dynamic behavior of lung surfactant.

Intravenous administration of perfluorocarbon (PFC) compounds can lead to pulmonary hyperinflation and respiratory distress in some mammals. This study was designed to quantify the effects of two PFC emulsions on the dynamic behavior of lung surfactant and to demonstrate that PFC is retained in the liquid lining the lung. New Zealand White rabbits received isotonic saline (3 ml/kg), Fluosol (15 ml/kg) or Oxygent (90% perfluorooctyl-bromide emulsion, 3 ml/kg). After seven days we euthanized the animals and lavaged the lungs. Surface tension-surface area relationships (sigma-A loops) were measured with the lavage fluid placed in a Wilhelmy plate-oscillating bellows apparatus. Loop hysteresis area after Fluosol administration was 334 +/- 92 dyne-cm, significantly greater than after saline (203 +/- 36 dyne-cm) but not Oxygent (274 +/- 66 dyne-cm). Loop hysteresis slope was higher with Oxygent (0.8 +/- 0.4 dyne/cm3) than after saline (0.6 +/- 0.3 dyne/cm3) or Fluosol (0.5 +/- 0.1 dyne/cm3). 282 MHz 19F NMR spectral analysis demonstrates that both PFCs tested appear only in the extracellular fraction of the lavage fluid. These results show that pulmonary elimination of intravascular PFC leads to PFC presence in the liquid lining the airways where it alters surfactant dynamic mechanical behavior.

Perfluorocarbon induced alterations in pulmonary mechanics.

Perfluorocarbon (PFC) compounds induce pulmonary hyperinflation and respiratory distress in some animals following intravenous administration. This study was designed to quantify the effects of two PFC emulsions on lung volumes and compliance and to identify the mechanism of pulmonary hyperinflation. New Zealand White rabbits received isotonic saline (3 ml/kg), Fluosol (15 ml/kg) or Oxygent (90% perfluorooctyl-bromide emulsion, 3 ml/kg). After seven days we measured functional residual capacity, vital capacity, lung compliance and thoracic gas volume. Gross and microscopic histologic examination of the lungs was performed. Functional residual capacity after Fluosol administration was 16.0 +/- 4.0 ml/kg, significantly greater than after saline (3.4 +/- 1.0 ml/kg) or Oxygent (4.0 +/- 1.4 ml/kg). Vital capacity was lower with Fluosol (30 +/- 5.0 ml/kg) than after saline (37 +/- 3.0 ml/kg) or Oxygent (37 +/- 2.0 ml/kg). Thoracic gas volume increased from 9 +/- 1.0 ml/kg (saline) to 16 +/- 13 ml/kg (Oxygent) and 33 +/- 7.0 ml/kg (Fluosol). Lung compliance was the same after saline (1.6 +/- 0.5 ml.cm H2O-1.kg-1) and Oxygent (1.5 +/- 0.3 ml.cm H2O-1.kg-1) but lower after Fluosol (0.9 +/- 0.1 ml.cm H2O-1.kg-1). Gross pathology demonstrated foam exudation from airways of animals receiving PFCs and intra-alveolar foam was identified by light microscopy. These results show intra-airway foam formation causes gas trapping and shifts tidal breathing to a less compliant region of the pressure-volume curve.

Regional variation in capillary hemodynamics in the cat retina.

PURPOSE: The behavior of the retinal microcirculation and its role in the progression of ocular disease is of considerable interest, yet few details are known about the flow of blood through the capillary networks of the retina. Although retinal vessels may be viewed through the pupil using standard optics, the optical limitations of the cornea and lens prevent the resolution of retinal features smaller than approximately 10 microns in size. Because red blood cells are smaller than this, fluorescent techniques such as angiography, specific cell labeling, and fluorescein-encapsulated liposomes have typically been used to observe the retinal microcirculation in vivo. Here the authors report a study of in vivo retinal capillary hemodynamics using white light GRadient INdex of refraction (GRIN) lens endoscopy. METHODS: GRIN lens endoscopy and robotic manipulation were used to directly observe and record the motion of erythrocytes within retinal capillary networks. Video images from the endoscope were analyzed to determine the regional variation of erythrocyte velocity and normalized optical density (an index of relative capillary hematocrit) in the superficial retinal capillaries of the cat. RESULTS: A significant decrease in mean retinal capillary velocity coupled with a corresponding increase in red blood cell density was observed in peripheral regions of the retina when compared with regions of the retina near the optic disc. Stasis or intermittent flow was not observed in the unstained retina, nor were capillaries noted that contained only plasma. CONCLUSIONS: Quantification of the bloodflow in retinal microcirculation was possible using GRIN lens endoscopy and showed significant regional heterogeneity in the cat retina.

Mechanical forces alter growth factor release by pleural mesothelial cells.

The mesothelial cells that form the visceral pleura of the lung are subjected to physical forces such as stretch due to lung expansion and fluid shear stress due to the sliding motion of the lung against the chest wall. In this study, the effect of mechanical forces on the production of growth factors by mesothelial cells was investigated. Rat visceral pleura mesothelial (RVPM) cells were exposed to fluid shear stress by perfusing a column of cell-covered beads. RVPM cells grown on a silicone elastomer were subjected to cyclic strain by applying an oscillating vacuum to the bottom of the wells using the Flexercell apparatus. Fluid shear stress (5.2-15.7 dyn/cm2) stimulated the release of endothelin-1 (ET-1) by RVPM cells two- to fivefold over static cells. ET-1 secretion by RVPM cells was also stimulated approximately twofold by cyclic stretch (20% maximum strain, 30 cycles/min). RVPM cells released significant levels of platelet-derived growth factor (PDGF), but there was no effect of either shear stress or cyclic strain on PDGF release. These results suggest that the production of growth factors by pleural mesothelial cells is regulated in part by physical forces.

Toward robot-assisted vascular microsurgery in the retina.

BACKGROUND: Experimental protocol in our laboratory routinely requires the precise placement of instruments at, or near, the retina. Although manipulators for placing an instrument within the eye presently exist, none of the designs were satisfactory due to limitations on size, accuracy and operability. To overcome these limitations, we have developed a novel six degree of freedom manipulator designed specifically for retinal microsurgery. METHODS: The manipulator is parallel in structure and provides submicrometer positioning of an instrument within the constrained environment of the eye. The position of an instrument attached to the manipulator is commanded by the operator using a hand-held trackball. A computer controller interprets the trackball input and moves the manipulator in an intuitive manner according to mathematically constrained modes of operation. RESULTS: Over 50 retinal vessels in the live, anesthetized cat have been successfully cannulated for pressure measurement and drug injection using the described manipulator and micropuncture techniques. The targeted vessels ranged in internal diameter from 20 to 130 microns. CONCLUSION: This device has applications in microsurgery where tremor and fatigue limit the performance of an unaided hand and where mechanically constrained manipulators are inappropriate due to size and operative constraints.

The effect of acute experimental retinal vein occlusion on cat retinal vein pressures.

PURPOSE: Retinal ischemic damage associated with retinal vein occlusion is exacerbated by fluid extravasation and hemorrhage, which may be caused by increased permeability, elevated intravascular pressure, or both. Direct measurement of the retinal vein pressure in the cat after acute experimental retinal vein occlusion may define the role of intravascular pressures in fluid extravasation associated with this condition. METHODS: Intravenous retinal pressure measurements were obtained using a micropipette connected to a servonull device and positioned by a robot micromanipulator, while a major retinal vein near the optic disc was occluded by argon laser radiation delivered through an optical fiber positioned by a manual micromanipulator. After occlusion, retinal vein pressures were measured on both sides of the occlusion site at a controlled intraocular pressure of 20 mm Hg. RESULTS: Upstream of the occlusion site, the retinal vein pressures were not greatly elevated, although they were significantly different from controls. Downstream vein pressures were significantly lower than controls, but vascular collapse near the optic nerve was not observed. CONCLUSIONS: In retinal vein occlusion, venous pressures in a segmental retinal circulatory bed are not substantially elevated, thus implying the presence of a pressure-release mechanism and implicating vascular damage for the increased transvascular fluid flux. The lack of vascular collapse downstream of the occlusion site suggests collateral communication before a large intraocular pressure-dependent resistance segment that lies between the intraocular and extraocular vessels.

Control mechanisms of retinal vein pressure: evaluation in the cat using an in situ infusion micropipette.

OBJECTIVE: Previous work in our laboratory has shown that retinal vein pressure is significantly greater than intraocular pressure in the cat. Our purpose here is to evaluate the potential of an active mechanism being responsible for the control of vein pressure in the cat retina. METHODS: A double-lumen, concentric barrel micropipette assembly designed for operation within the eye of a spontaneously breathing anesthetized cat was developed. The micropipette, initially filled with hypertonic saline for use with a servonull pressure measuring system, was used to determine intravascular pressure. With the tip of the micropipette still situated in the lumen of the vessel the solution in the micropipette was exchanged for papaverine, a potent smooth muscle dilator. Following vasodilation in the distal portion of a major retinal vein, the papaverine was exchanged for hypertonic saline and intravascular pressures were measured in the dilated vessel. RESULTS: Microinjection of papaverine caused visible dilation of the major retinal vein downstream from the site of microinjection. This indicates a relaxation of the smooth muscle yet papaverine caused no change in measured retinal vein pressure. CONCLUSIONS: Vein pressure in the cat retina is maintained above intraocular pressure by mechanisms other than active control. A passive high-resistance segment resides somewhere within the optic nerve possibly within the lamina cribosa or prelaminar region.

Shear stress alters pleural mesothelial cell permeability in culture.

The sliding motion of the lung against the chest wall creates a shear stress in the pleural space, which can be as high as 60 dyn/cm2, depending on the respiration rate. Such shear stresses may affect the mesothelial cells that line the pleural space on the lung (visceral pleura) and chest wall (parietal pleura). When exposed to shear stress (17 dyn/cm2) in a parallel-plate flow chamber for 22 h, rat visceral pleura mesothelial cells were not altered morphologically and did not align in the direction of flow, in contrast to the shape changes observed for bovine aortic endothelial cells. By using mesothelial cells cultured on porous microcarrier beads, we measured the permeability of the cells at different flows in a cell-column chromatography assay. The permeabilities to sodium fluorescein and cyanocobalamin increased from 8.2 +/- 1.0 and 7.8 +/- 0.7 x 10(-5) cm/s to 22.5 +/- 1.2 and 21.8 +/- 3.0 x 10(-5) cm/s, respectively, when the flow was increased from 0.9 to 3.5 ml/min (corresponding to average shear stresses of 4.7-18.4 dyn/cm2). The permeabilities returned to baseline values when the flow was reduced. Cytochalasin D stimulated an increase in permeability that was not augmented by a subsequent increase in shear stress. These results suggest that the barrier function of mesothelial cells is responsive to changes in fluid shear stress.

A buoyancy-driven squeeze-film model of intrapleural fluid dynamics: basic concepts.

We propose a new model of pleural liquid mechanics in the apneic animal based on the observation that the lung, in its position within the serous fluid of the chest, is a buoyant object with few physical connections to the chest. The buoyancy force due to the lung's low density causes the lung to rise within the chest, which in turn causes fluid to be squeezed out from the regions above the lung. The result is the transient component of the downward flow of intrapleural liquid and the less-than-hydrostatic vertical intrapleural pressure gradient observed by other investigators. For the purposes of mathematical simplicity, we have modeled the lung and chest wall of a horizontal animal as a pair of concentric cylinders separated by a narrow gap representing the pleural space. In this first version of the model, we treat the pleurae of the lung and chest wall as impermeable rigid boundaries, but despite these limitations, our mathematical analysis agrees with observations from a number of groups and explains the flow direction from top to bottom as well as the reported changes in vertical pressure gradient in response to a change in body orientation.

The influence of elevated intraocular pressure on vascular pressures in the cat retina.

PURPOSE: Elevated intraocular pressure is known to reduce retinal blood flow, although the effect of intraocular pressure on retinal vascular pressures is unknown. Direct measurements of intravascular pressures were taken in the cat retina at various intraocular pressures. METHODS: Micropipettes of 2- to 3-microns tip diameter were used in conjunction with a servonull pressure-measuring system to determine retinal intravascular pressures in supine anesthetized cats. Pressures in large (80 to 120 microns diameter) vessels near the optic disc were measured over a wide range of intraocular pressures. RESULTS: Measurements show that retinal artery pressure depends on both intraocular pressure and mean systemic blood pressure, and that retinal vein pressure is determined by, but generally is different from, intraocular pressure, with no significant correlation to mean systemic blood pressure. Empirical equations are presented that predict statistically significant retinal artery, vein, and microvascular perfusion pressures. CONCLUSIONS: Intraocular pressure is an important determinant of the microvascular perfusion pressure in the retina of the cat, particularly at low mean systemic blood pressure. It is also apparent that retinal vein pressure is always greater than intraocular pressure, which implies the existence of a high-resistance extraretinal segment of the retinal vein. The results suggest mechanisms for the loss of visual function in glaucoma and other retinal circulatory disorders.

In vivo micropuncture of retinal vessels.

Micropuncture has proven to be a valuable tool for the local study of vascular parameters in many organ systems; however, it has not been applied to the study of the circulation of the retina. We report here our extension of micropuncture techniques [4] to use in the intact retina of the anesthetized cat. We use extremely sharp micropipettes with tip sizes much smaller than the diameter of erythrocytes to avoid hemorrhage. The micropipette is held by a microdrive which in turn is mounted on a precision goniometric micromanipulator. We micropuncture retinal arteries and veins with diameters ranging from 20 to 130 microns with no apparent damage to the vessel wall and no observed hemorrhage. During micropuncture we routinely inject nanoliter quantities of dyed saline, which we observe flowing in a plume from the micropipette tip within the lumen of the vessel. Micropuncture techniques may be used in the laboratory to study retinal autoregulatory mechanisms by microinfusion of vasoactive substances and by measuring blood pressure in retinal microvessels. In the clinic micropuncture may be useful for treating disorders such as retinal vascular occlusion.

Effect of vascular pressure on interstitial pressures in the isolated dog lung.

We report the first direct measurements of the effect of pulmonary vascular pressures on perialveolar interstitial pressures. In seven experiments we varied the intravascular pressure (Pvas) in isolated dog lungs held at constant airway pressure (PA). By the micropuncture servo-null technique, we recorded perialveolar interstitial pressures with respect to pleural pressure (0 cmH2O) at the alveolar junctions (Pjct) and in microvascular adventitia (Padv). At PA = 7 cmH2O, increase from 5 to 15 cmH2O did not affect Pjct, although it decreased Padv by 1.2 +/- 0.4 cmH2O. The Pjct-Padv gradient increased by 77%. Increasing Pvas to 25 cmH2O had no further effect on either interstitial pressure. In four experiments we also determined interstitial pressure in the hilum (Phil). When Pvas was increased from 5 to 15 cmH2O, Phil increased by 4.5 +/- 0.9 cmH2O. Further elevation of Pvas to 25 cmH2O increased Phil further by 2.4 +/- 0.4 cmH2O. At PA = 15 cmH2O, all interstitial pressures decreased, but their responses to Pvas were similar. We conclude that increase of Pvas 1) increases Phil but not perialveolar interstitial pressures and 2) increases the perialveolar interstitial pressure gradient, which may promote local liquid clearance.

Direct measurement of retinal microvascular pressures in the live, anesthetized cat.

Microvascular pressures in retinal circulation were measured in living, anesthetized cat by the servonull micropuncture technique. Of the total vascular pressure drop 32% was in the segment proximal to the large retinal arteries when intraocular pressure was set at 10 mm Hg and decreased to 27% when intraocular pressure was increased to 20 mm Hg. The pressure in the retinal veins was substantially (> 7 mm Hg) greater than intraocular pressure at both values of intraocular pressure. There was negligible pressure drop within venous segment; however, the pressure drop from the retinal veins to the systemic veins was large and cannot be explained on the basis of a venous waterfall within the retina.