Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy.

Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung.

Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal cantilever tips.

Atomic force microscopy (AFM) allows the acquisition of high-resolution images and the measurement of mechanical properties of living cells under physiological conditions. AFM cantilevers with blunted pyramidal tips are commonly used to obtain images of living cells. Measurement of mechanical properties with these tips requires a contact model that takes into account their blunted geometry. The aim of this work was to develop a contact model of a blunted pyramidal tip and to assess the suitability of pyramidal tips for probing mechanical properties of soft gels and living cells. We developed a contact model of a blunted pyramidal tip indenting an elastic half-space. We measured Young's modulus (E) and the complex shear modulus (G*= G' +i G" ) of agarose gels and A549 alveolar epithelial cells with pyramidal tips and compared them with those obtained with spherical tips. The gels exhibited an elastic behavior with almost coincident loading and unloading force curves and negligible values of G". E fell sharply with indentation up to approximately 300 nm , showing a linear regime for deeper indentations. A similar indentation dependence of E with twofold lower values at the linear regime was obtained with the spherical tip fitted with Hertz's model. The dependence of E on indentation in cells paralleled that found in gels. Cells exhibited viscoelastic behavior with G"/G' approximately 1/4 . Pyramidal tips commonly used for AFM imaging are suitable for probing mechanical properties of soft gels and living cells.

Animal model of unilateral ventilator-induced lung injury.

OBJECTIVE: To design, implement, and test a selective lung ventilator for setting a rat model of unilateral ventilator-induced lung injury (VILI). DESIGN AND SETTING: Interventional animal study in a university laboratory for animal research. SUBJECTS: Anesthetized and paralyzed male Wistar rats. INTERVENTIONS: A selective ventilator designed to apply varying tidal volume, PEEP, and breathing gas to each lung of the rat was implemented and evaluated. Five control animals were ventilated at 7 ml/kg (3.5 ml/kg each lung). Unilateral VILI was induced in six animals subjected to selective ventilation (3.5 ml/kg in one lung and 15 ml/kg in the other lung). After 3 h of ventilation the animals were killed and the lungs excised. MEASUREMENTS AND RESULTS: Lung edema was assessed by means of the ratio between wet and dry lung weights. No significant differences were found in lungs of control animals (5.16+/-0.22 and 4.96+/-0.25), but the W/D ratio in the over ventilated lung (8.98+/-3.80) was significantly greater than that in the normally ventilated lung (4.76+/-0.15), indicating selective induction of lung edema by over stretch. CONCLUSIONS: This selective ventilator can be implemented into a rat model of unilateral VILI to gain further insight into the mechanisms of pulmonary injury induced by different ventilatory strategies.

Collapsible upper airway segment to study the obstructive sleep apnea/hypopnea syndrome in rats.

Animal models have been used to study the pathophysiology of the obstructive sleep apnea/hypopnea syndrome (SAHS). Nevertheless, in none of the models described to date have the animals been subjected to the different patterns of upper airway obstructive events (apneas, hypopneas, and inspiratory flow limitation) characterizing SAHS. Our aim was to devise and test a computer-controlled collapsible upper airway segment applicable to rats and able to realistically mimic obstructive SAHS events. The collapsible segment (total volume <2 cm(3) and a dead space of approximately 0.25 cm(3)) consisted of a Starling resistor based on a latex membrane subjected to an external pressure applied by a computer-controlled pressure source. The collapsible segment was tested in eight anaesthetized and tracheostomized rats. The upper airway segment allowed us to induce obstructive apneas and hypopneas with flow and inspiratory effort waveforms similar to the ones observed in patients with SAHS. This collapsible upper airway segment may be a useful tool to implement a rat model of SAHS.