Real-time imaging of cellular and sub-cellular dynamics in vascularized organs requires image-resolution, image-registration, and demonstrably intact physiology to be simultaneously optimized. This problem is particularly pronounced in the lung in which cells may transit at speeds > 1 mm/sec, and in which normal respiration results in large-scale tissue movements that prevent image registration. Here, we report video-rate, two-photon imaging of a physiologically intact preparation of the mouse lung that is at once stabilizing and non-disruptive. The application of our method provides evidence for differential trapping of T cells and neutrophils in mouse pulmonary capillaries and enables observation of neutrophil mobilization and dynamic vascular leak in response to stretch and inflammatory models of lung injury in mice. The system permits physiological measurement of motility rates of > 1 mm/sec, observation of detailed cellular morphology, and could be applied to other organs and tissues while maintaining intact physiology.

Regional pulmonary blood flow in dogs under zone 3 conditions was measured in supine and prone postures to evaluate the linear gravitational model of perfusion distribution. Flow to regions of lung that were 1.9 cm3 in volume was determined by injection of radiolabeled microspheres in both postures. There was marked perfusion heterogeneity within isogravitational planes (coefficient of variation = 42.5%) as well as within gravitational planes (coefficient of variation = 44.2 and 39.2% in supine and prone postures, respectively; P = 0.02). On average, vertical height explained only 5.8 and 2.4% of the flow variability in the supine and prone postures, respectively. Whereas the gravitational model predicts that regional flows should be negatively correlated when measured in supine and prone postures, flows in the two postures were positively correlated, with an r2 of 0.708 +/- 0.050. Regional perfusion as a function of distance from the center of a lung explained 13.4 and 10.8% of the flow variability in the supine and prone postures, respectively. A linear combination of vertical height and radial distance from the centers of each lung provided a better-fitting model but still explained only 20.0 and 12.0% of the flow variability in the supine and prone postures, respectively. The entire lung was searched for a region of contiguous lung pieces (22.8 cm3) with high flow. Such a region was found in the dorsal area of the lower lobes in six of seven animals, and flow to this region was independent of posture. Under zone 3 conditions, neither gravity nor radial location is the principal determinant of regional perfusion distribution in supine and prone dogs.

SummaryInhalation of antigen in immunized mice induces an infiltration of eosinophils into the airways and increased bronchial hyperreactivity as are observed in human asthma. We employed a model of late-phase allergic pulmonary inflammation in mice to address the role ofleukotrienes (LT) in mediating airway eosinophilia and hyperreactivity to methacholine. Allergen intranasal challenge in OVA-sensitized mice induced LTB4 and LTC 4 release into the airspace, widespread mucus occlusion of the airways, leukocytic infiltration of the airway tissue and bronchoalveolar lavage fluid that was predominantly eosinophils, and bronchial hyperreactivity to methacholine. Specific inhibitors of 5-1ipoxygenase and 5-1ipoxygenase-activating protein (FLAP) blocked airway mucus release and infiltration by eosinophils indicating a key role for leukotrienes in these features of allergic pulmonary inflammation. The role of leukotrienes or eosinophils in mediating airway hyperresponsiveness to aeroailergen could not be established, however, in this murine model.

The mechanism by which oxygenation improves when patients with ARDS are turned from supine to prone position is not known. From results of our previous studies we reasoned that (1) when supine, in the setting of lung injury, transpulmonary pressure will be less than airway opening pressure and (2) atelectasis will develop preferentially in dorsal lung areas, and (3) both ventilation and ventilation/perfusion ratios would improve in these regions on turning prone. To study this directly, we measured regional ventilation and perfusion using 81mKr and 99mTc-MAA, respectively, and single photon emission computed tomography, both prone and supine, in four control animals and four given oleic acid. After oleic acid, the prone position improved (1) oxygenation (mean +/- SD PaO2 = 140 +/- 112 versus 453 +/- 54 mm Hg), (2) median ventilation/perfusion ratios (0.77 versus 0.95), (3) ventilation/perfusion heterogeneity (coefficient of variation 86 +/- 15 versus 61 +/- 6), and (4) the gravitational ventilation/perfusion gradient (dependent to non-dependent slopes of 0.22 versus -0.02, all p < 0.05). The prone position generates a transpulmonary pressure sufficient to exceed airway opening pressure in dorsal lung regions, i.e., in regions where atelectasis, shunt, and ventilation/perfusion heterogeneity are most severe, without adversely affecting ventral lung regions.

We used various ovalbumin sensitization and challenge protocols to determine the importance of the route of allergen administration and the genetic background in modulating the physiologic, inflammatory, and immunologic features characteristic of allergen-induced asthma. In BALB/c mice, induction of maximal airway hyperresponsiveness and airspace eosinophilia required administration of ovalbumin by both the intraperitoneal and the intranasal routes (combination protocol), whereas intraperitoneal immunization alone resulted in maximal ovalbumin-specific IgE plasma levels. Thus, a systemic immune response to allergen, in addition to, or independent of IgE production, as well as local allergen challenge were necessary for maximal induction of pulmonary disease. BALB/c mice treated with ovalbumin by the combination protocol had increased Th2-type cytokine mRNA levels in bronchial lymph node tissue compared with control mice. In contrast, C57BL/6 mice treated with ovalbumin by the combination protocol had significantly decreased responses compared with BALB/c mice for all parameters of allergic pulmonary disease examined, with the exception of airspace eosinophilia. Genetic background has a striking and selective effect on the phenotype of murine allergic pulmonary disease. Further analysis of this murine model should be useful in helping define the critical pathogenetic events in allergen-induced asthma.

To investigate whether hypercapnic acidosis protects against ventilator-induced lung injury (VILI) in vivo, we subjected 12 anesthetized, paralyzed rabbits to high tidal volume ventilation (25 cc/kg) at 32 breaths per minute and zero positive end-expiratory pressure for 4 hours. Each rabbit was randomized to receive either an FI(CO(2)) to achieve eucapnia (Pa(CO(2)) approximately 40 mm Hg; n = 6) or hypercapnic acidosis (Pa(CO(2)) 80-100 mm Hg; n = 6). Injury was assessed by measuring differences between the two groups' respiratory mechanics, gas exchange, wet:dry weight, bronchoalveolar lavage fluid protein concentration and cell count, and injury score. The eucapnic group showed significantly higher plateau pressures (27.0 +/- 2.5 versus 20.9 +/- 3.0; p = 0.016), change in Pa(O(2)) (165.2 +/- 19.4 versus 77.3 +/- 87.9 mm Hg; p = 0.02), wet:dry weight (9.7 +/- 2.3 versus 6.6 +/- 1.8; p = 0.04), bronchoalveolar lavage protein concentration (1,350 +/- 228 versus 656 +/- 511 micro g/ml; p = 0.03), cell count (6.86 x 10(5) +/- 0.18 x 10(5) versus 2.84 x 10(5) +/- 0.28 x 10(5) nucleated cells/ml; p = 0.021), and injury score (7.0 +/- 3.3 versus 0.7 +/- 0.9; p < 0.0001). We conclude that hypercapnic acidosis is protective against VILI in this model.

Oxygenation improves in patients with adult respiratory distress syndrome and in animals with oleic acid-induced lung injury when they are turned from the supine to the prone position. Dependent and nondependent pleural pressures (Ppl) were measured in six pigs ventilated in the supine and prone positions before and after volume infusion (VI). Before VI the mean +/- SEM AaPO2 difference was 26 +/- 8 mm Hg when the animals were supine and 10 +/- 2 mm Hg when they were prone (p > 0.05). After VI the AaPO2 was 64 +/- 6 mm Hg when the animals were supine (p < 0.05) and 43 +/- 7 mm Hg when they were prone (p < 0.05). VI increased the Ppl gradient from 0.53 +/- 0.1 to 0.71 +/- 0.1 cm H2O/cm when the animals were supine (p < 0.05) and from 0.17 +/- 0.1 to 0.27 +/- 0.1 cm H2O/cm when they were prone (p < 0.05). Dependent Ppl at FRC was much less positive when the animals were prone versus supine (0.9 +/- 0.3 versus 3.0 +/- 0.5 cm H2O, p < 0.05), suggesting that the airways in these dependent regions would narrow and/or close and that ventilation to these regions would diminish as a result of VI.

Exhaled acetone is measured to estimate exposure or monitor diabetes and congestive heart failure. Interpreting this measurement depends critically on where acetone exchanges in the lung. Health professionals assume exhaled acetone originates from alveolar gas exchange, but experimental data and theoretical predictions suggest that acetone comes predominantly from airway gas exchange. We measured endogenous acetone in the exhaled breath to evaluate acetone exchange in the lung. The acetone concentration in the exhalate of healthy human subjects was measured dynamically with a quadrupole mass spectrometer and was plotted against exhaled volume. Each subject performed a series of breathing maneuvers in which the steady exhaled flow rate was the only variable. Acetone phase III had a positive slope (0.054+/-0.016 liter-1) that was statistically independent of flow rate. Exhaled acetone concentration was normalized by acetone concentration in the alveolar air, as estimated by isothermal rebreathing. Acetone concentration in the rebreathed breath ranged from 0.8 to 2.0 parts per million. Normalized end-exhaled acetone concentration was dependent on flow and was 0.79+/-0.04 and 0.85+/-0.04 for the slow and fast exhalation rates, respectively. A mathematical model of airway and alveolar gas exchange was used to evaluate acetone transport in the lung. By doubling the connective tissue (epithelium+mucosal tissue) thickness, this model predicted accurately (R2=0.94+/-0.05) the experimentally measured expirograms and demonstrated that most acetone exchange occurred in the airways of the lung. Therefore, assays using exhaled acetone measurements need to be reevaluated because they may underestimate blood levels.