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.

This review provides an overview of the relationship between ventilation/perfusion ratios and gas exchange in the lung, emphasising basic concepts and relating them to clinical scenarios. For each gas exchanging unit, the alveolar and effluent blood partial pressures of oxygen and carbon dioxide (PO 2 and PCO 2 ) are determined by the ratio of alveolar ventilation to blood flow (V9A/Q9) for each unit. Shunt and low V9A/Q9 regions are two examples of V9A/Q9 mismatch and are the most frequent causes of hypoxaemia. Diffusion limitation, hypoventilation and low inspired PO 2 cause hypoxaemia, even in the absence of V9A/Q9 mismatch. In contrast to other causes, hypoxaemia due to shunt responds poorly to supplemental oxygen. Gas exchanging units with little or no blood flow (high V9A/Q9 regions) result in alveolar dead space and increased wasted ventilation, i.e. less efficient carbon dioxide removal. Because of the respiratory drive to maintain a normal arterial PCO 2 , the most frequent result of wasted ventilation is increased minute ventilation and work of breathing, not hypercapnia. Calculations of alveolar-arterial oxygen tension difference, venous admixture and wasted ventilation provide quantitative estimates of the effect of V9A/Q9 mismatch on gas exchange. The types of V9A/Q9 mismatch causing impaired gas exchange vary characteristically with different lung diseases. @ERSpublications A review of ventilation-perfusion relationships and gas exchange, basic concepts and their relation to clinical cases

Estimations of dog lung, pig heart, and pig kidney regional perfusion by use of fluorescent-labeled microspheres were compared with measurements obtained with standard radiolabeled microspheres. Pairs of radio- and fluorescent-labeled microspheres (15 microns diam, 6 colors) were injected into a central vein of a supine anesthetized dog and the left ventricle of three supine anesthetized pigs while reference blood samples were simultaneously withdrawn from a femoral artery in the pigs. The lungs were cubed into approximately 2 cm3 pieces (n = 1,510). Each pig heart and kidney was cubed into approximately 1-g pieces (total n = 192 and 120, respectively). The radioactivity of each organ piece and reference blood sample was determined using a scintillation counter with count rates corrected for decay, background, and spillover. Tissue samples and reference blood samples were digested with KOH and filtered and the fluorescent dye was extracted with a solvent, or the dye was extracted from lung tissue without filtering. The fluorescence of each sample was determined for each color by use of an automated spectrophotometer. Perfusion was calculated for each organ piece from both the radioactivity and fluorescence. Correlation between flow determined by radio- and fluorescent-labeled microspheres was as follows: r = 0.96 +/- 0.01 (SD) (lung, filtered, n = 588), r = 0.99 +/- 0.00 (lung, nonfiltered, n = 710), r = 0.95 +/- 0.02 (heart, filtered), and r = 0.96 +/- 0.02 (kidney, filtered). Compared with colored microspheres, methods for quantitating fluorescent-labeled microspheres are more sensitive, less labor intensive, and less expensive. Fluorescent-labeled microspheres provide a new nonradioactive method for single and repeated measurement of regional organ perfusion.

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Previous animal studies have identified a role for activation of innate immunity in the pathogenesis of ventilator-associated lung injury. These studies have used large tidal volume ventilation to study the effect of alveolar overdistension on induction of inflammatory pathways. We hypothesized an alternative mechanism for the pathogenesis of lung injury in which moderate tidal volume ventilation does not independently cause clinical inflammation but rather interacts with innate immune activation by bacterial products, resulting in an enhanced inflammatory response. We measured cytokine expression and lung injury in normal and lipopolysaccharide (LPS)-treated anesthetized rabbits randomized to either spontaneous respiration or mechanical ventilation. Outcome parameters were analyzed by two-way factorial analysis of variance to identify synergism between ventilation and systemic LPS. Mechanical ventilation alone resulted in minimal cytokine expression in the lung but did enhance LPS-induced expression of tumor necrosis factor-alpha, the CXC chemokines interleukin-8 and growth-related protein-alpha, and the CC chemokine monocyte chemoattractant protein-1. Increased mRNA expression and activation of the transcription factors nuclear factor-kappaB and activator protein-1 accompanied the cytokine responses. We conclude that moderate volume ventilation strategies augment the innate immune response to bacterial products in the lung and may play a role in the development of acute lung injury in patients with sepsis.

The heterogeneity of pulmonary blood flow was examined using a fractal analytic procedure, and the results were compared with the traditional gravitational model of flow distribution. 99mTc-labeled macroaggregate was injected intravenously at functional residual capacity in six supine anesthetized dogs. The lungs were fixed in situ and sliced in transverse sections. The slices were imaged on a planar gamma camera, and a three-dimensional array of blood flow measurements was reconstructed for each lung. Fractal analysis was used to examine the spatial heterogeneity or RDs (relative dispersion = SD/mean) as a function of the number of pieces into which the flow array was subdivided. RDs was fractal and could be characterized by a fractal dimension (Ds) of 1.09 +/- 0.02, where a Ds of 1.0 reflects homogeneous flow and 1.5 indicates a random flow distribution. The data fit the fractal model exceptionally well with an average r = 0.98. RDs was examined in gravitational and isogravitational planes and as expected was greatest in the gravitational direction. However, the difference was small, suggesting that gravitation plays a secondary role to an underlying process producing heterogeneity. Within the limits of resolution attained by this study (piece volumes greater than 0.25 cm3), the heterogeneity of pulmonary blood flow is well characterized by a fractal model.

Computational fluid dynamics (CFD) models are useful for predicting site-specific dosimetry of airborne materials in the respiratory tract and elucidating the importance of species differences in anatomy, physiology, and breathing patterns. We improved the imaging and model development methods to the point where CFD models for the rat, monkey, and human now encompass airways from the nose or mouth to the lung. A total of 1272, 2172, and 135 pulmonary airways representing 17±7, 19±9, or 9±2 airway generations were included in the rat, monkey and human models, respectively. A CFD/physiologically based pharmacokinetic model previously developed for acrolein was adapted for these anatomically correct extended airway models. Model parameters were obtained from the literature or measured directly. Airflow and acrolein uptake patterns were determined under steady-state inhalation conditions to provide direct comparisons with prior data and nasal-only simulations. Results confirmed that regional uptake was sensitive to airway geometry, airflow rates, acrolein concentrations, air:tissue partition coefficients, tissue thickness, and the maximum rate of metabolism. Nasal extraction efficiencies were predicted to be greatest in the rat, followed by the monkey, and then the human. For both nasal and oral breathing modes in humans, higher uptake rates were predicted for lower tracheobronchial tissues than either the rat or monkey. These extended airway models provide a unique foundation for comparing material transport and site-specific tissue uptake across a significantly greater range of conducting airways in the rat, monkey, and human than prior CFD models.

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