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.     

Effect of viscosity on instilled perfluorocarbon distribution in rabbit lungs

The effect of viscosity on the distribution of perfluorocarbon instilled into the lungs for liquid ventilation was investigated. Perfluorocarbon (either perfluorodecalin or FC-3283) was instilled into the trachea during ventilation at a constant infusion rate of 40 ml/min and radiographic images were obtained at 30 frames/s. Image analysis was performed and the homogeneity index of the distribution was computed for images at the end of inspiration of each breath to evaluate the evolution of perfluorocarbon distribution during filling. The higher viscosity perfluorocarbon (perfluorodecalin) resulted in a more homogeneous distribution. This was attributed to perfluorodecalin's higher propensity to form liquid plugs in large airways and to those plugs leaving behind a thicker liquid layer as they propagated through the lungs.     

Distribution dynamics of perfluorocarbon delivery to the lungs: an intact rabbit model.

Motivated by the goal of understanding how to most homogeneously fill the lungs with perfluorocarbon for liquid ventilation, we investigate the transport of liquid instilled into the lungs using an intact rabbit model. Perfluorocarbon is instilled into the trachea of the ventilated animal. Radiographic images of the perfluorocarbon distribution are obtained at a rate of 30 frames/s during the filling process. Image analysis is used to quantify the liquid distribution (center of mass, spatial standard deviation, skewness, kurtosis, and indicators of homogeneity) as time progresses. We compare the distribution dynamics in supine animals to those in upright animals for three constant infusion rates of perfluorocarbon: 15, 40, and 60 ml/min. It is found that formation of liquid plugs in large airways, which is affected by posture and infusion rate, can result in a more homogeneous liquid distribution than gravity drainage alone. The supine posture resulted in more homogeneous filling of the lungs than did upright posture, in which the lungs tend to fill in the inferior regions first. Faster instillation of perfluorocarbon results in liquid plugs forming in large airways and, consequently, more uniform distribution of perfluorocarbon than slower instillation rates in the upright animals.     

Protective effect of lung inflation in reperfusion-induced lung microvascular injury

We used the isolated-perfused rat lung model to study the influence of pulmonary ventilation and surfactant instillation on the development of postreperfusion lung microvascular injury. We hypothesized that the state of lung inflation during ischemia contributes to the development of the injury during reperfusion. Pulmonary microvascular injury was assessed by continuously monitoring the wet lung weight and measuring the vessel wall (125)I-labeled albumin ((125)I-albumin) permeability-surface area product (PS). Sprague-Dawley rats (n = 24) were divided into one control group and five experimental groups (n = 4 rats per group). Control lungs were continuously ventilated with 20% O(2) and perfused for 120 min. All lung preparations were ventilated with 20% O(2) before the ischemia period and during the reperfusion period. The various groups differed only in the ventilatory gas mixtures used during the flow cessation: group I, ventilated with 20% O(2); group II, ventilated with 100% N(2); group III, lungs remained collapsed and unventilated; group IV, same as group III but pretreated with surfactant (4 ml/kg) instilled into the airway; and group V, same as group III but saline (4 ml/kg) was instilled into the airway. Control lungs remained isogravimetric with baseline (125)I-albumin PS value of 4.9 +/- 0.3 x 10(-3) ml x min(-1) x g wet lung wt(-1). Lung wet weight in group III increased by 1.45 +/- 0.35 g and albumin PS increased to 17.7 +/- 2.3 x 10(-3), indicating development of vascular injury during the reperfusion period. Lung wet weight and albumin PS did not increase in groups I and II, indicating that ventilation by either 20% O(2) or 100% N(2) prevented vascular injury. Pretreatment of collapsed lungs with surfactant before cessation of flow also prevented the vascular injury, whereas pretreatment with saline vehicle had no effect. These results indicate that the state of lung inflation during ischemia (irrespective of gas mixture used) and supplementation of surfactant prevent reperfusion-induced lung microvascular injury.     

A theoretical study of surfactant and liquid delivery into the lung

A computational study is presented for thetransport of liquids and insoluble surfactant through the lung airways,delivered from a source at the distal end of the trachea. Four distinct transport regimes are considered: 1)the instilled bolus may create a liquid plug that occludes the largeairways but is forced peripherally during mechanical ventilation;2) the bolus creates a deposited film on the airway walls, either from the liquid plug transport or fromdirect coating, that drains under the influence of gravity through thefirst few airway generations; 3) insmaller airways, surfactant species form a surface layer that spreadsdue to surface-tension gradients, i.e., Marangoni flows; and4) the surfactant finally reachesthe alveolar compartment where it is cleared according to first-orderkinetics. The time required for a quasi-steady-state transport processto evolve and for the subsequent delivery of the dose is predicted.Following fairly rapid transients, on the order of seconds,steady-state transport develops and is governed by the interaction ofMarangoni flow and alveolar kinetics. Total delivery time is ~24 hfor a typical first dose. Numerical solutions show that both transitand delivery times are strongly influenced by the strength of thepreexisting surfactant and the geometric properties of the airwaynetwork. Delivery times for follow-up doses can increase significantlyas the level of preexisting surfactant rises.

     

Topical application of phosphatidyl‐inositol‐3,5‐bisphosphate for acute lung injury in neonatal swine

Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro‐inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol‐3,5‐bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple‐hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte‐derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF‐β1) and interleukin‐6 as pro‐fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant “fortified” by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.     

Pulmonary and gastric surfactants. A comparison of the effect of surface requirements on function and phospholipid composition

Surfactant is present in the alveoli and conductive airways of mammalian lungs. The presence of surface active agents was, moreover, demonstrated for avian tubular lungs and for the stomach and intestine. As the surface characteristics of these organs differ from each other, their surfactants possess distinct biochemical and functional characteristics. In the stomach so-called 'gastric surfactant' forms a hydrophobic barrier to protect the mucosa against acid back-diffusion. For this purpose gastric mucosal cells secrete unsaturated phosphatidylcholines (PC), but no dipalmitoyl-PC (PC16:0/16:0). By contrast, surfactant from conductive airways, lung alveoli and tubular avian lungs contain PC16:0/16:0 as their main component in similar concentrations. Hence, there is no biochemical relation between gastric and pulmonary surfactant. Alveolar surfactant, being designed for preventing alveolar collapse under the highly dynamic conditions of an oscillating alveolus, easily reaches values of <5 mN/m upon cyclic compression. Surfactants from tubular air-exposed structures, however, like the conductive airways of mammalian lungs and the exclusively tubular avian lung, display inferior compressibility as they only reach minimal surface tension values of approximately 20 mN/m. Hence, the highly dynamic properties of alveolar surfactant do not apply for surfactants designed for air-liquid interfaces of tubular lung structures.     

The effect of diet-induced serum hypercholesterolemia on the surfactant system and the development of lung injury

Acute respiratory distress syndrome (ARDS) is a pulmonary disorder associated with alterations to the pulmonary surfactant system. Recent studies showed that supra-physiological levels of cholesterol in surfactant contribute to impaired function. Since cholesterol is incorporated into surfactant within the alveolar type II cells which derives its cholesterol from serum, it was hypothesized that serum hypercholesterolemia would predispose the host to the development of lung injury due to alterations of cholesterol content in the surfactant system.Wistar rats were randomized to a standard lab diet or a high cholesterol diet for 17–20 days. Animals were then exposed to one of three models of lung injury: i) acid aspiration ii) ventilation induced lung injury, and iii) surfactant depletion. Following physiological monitoring, lungs were lavaged to obtain and analyze the surfactant system.The physiological results showed there was no effect of the high cholesterol diet on the severity of lung injury in any of the three models of injury. There was also no effect of the diet on surfactant cholesterol composition. Rats fed a high cholesterol diet had a significant impairment in surface tension reducing capabilities of isolated surfactant compared to those fed a standard diet exposed to the surfactant depletion injury. In addition, only rats that were exposed to ventilation induced lung injury had elevated levels of surfactant associated cholesterol compared to non-injured rats.It is concluded that serum hypercholesterolemia does not predispose rats to altered surfactant cholesterol composition or to lung injury. Elevated cholesterol within surfactant may be a marker for ventilation induced lung damage.     

Respiratory movements alter the generation of prostacyclin and thromboxane A2 in isolated rat lungs: The influence of arachidonic acid-pathway inhibitors on the ratio between pulmonary prostacyclin and thromboxane A2

The influence of hyperventilation on the spontaneous generation of prostacyclin and thromboxane A₂ by isolated rat lungs was studied. Both prostacyclin and thromboxane A₂, as measured by RIA of their stable end-products, 6-oxo-PGF_1α and TXB₂ respectively, were continuously released into the perfusate. However, the concentration of prostacyclin in the perfusate was higher than thromboxane A₂. Under normal ventilation at a rate 40–50 breaths/min, the ratio between these two compounds was 5:1. Increasing the rate of respiration to 100 breaths/min preferentially stimulated the release of prostacyclin. During hyperventilation, the ratio between 6-oxo-PGF_1α and TXB₂ was 12:1. Aspirin and indomethacin suppressed both basal and hyperventilation-stimulated release of prostacyclin and thromboxane A₂. Hydroperoxy-fatty acids and tranylcypromine inhibited only the release of prostacyclin but did not affect the generation of thromboxane A₂. Our findings confirm that the lung generates prostacyclin predominantly, and provide direct evidence that respiratory movements are involved in generation of pulmonary prostacyclin and thromboxane A₂.     

Enhancement of the endotoxin recognition pathway by ventilation with a large tidal volume in rabbits

Ventilation with a small tidal volume (V(t)) is associated with better clinical outcomes than with a large V(t), particularly in critical settings, including acute lung injury. To determine whether V(t) influences the lipopolysaccaharide (LPS) recognition pathway, we studied CD14 expression in rabbit lungs and the release of TNF-alpha by cultured alveolar macrophages after 240 min of ventilation with a large (20 ml/kg) vs. a small (5 ml/kg) V(t). We also applied small or large V(t) to lungs instilled with 50 microg/kg of LPS. The alveolar macrophages collected after large V(t) ventilation revealed a 20-fold increase in LPS-induced TNF-alpha release compared with those collected after small V(t) ventilation, whereas TNF-alpha was undetectable without LPS stimulation. In animals ventilated with a large V(t), the expression of CD14 mRNA in whole lung homogenates and the expression of CD14 protein on alveolar macrophages, assessed by immunohistochemistry, were both significantly increased in the absence of LPS stimulation. A large V(t) applied to LPS-instilled lungs increased the pulmonary albumin permeability and TNF-alpha release into the plasma. These results suggest that mechanical stress caused by a large V(t) sensitizes the lungs to endotoxin, a phenomenon that may occur partially via the upregulation of CD14.     

Alterations to surfactant precede physiological deterioration during high tidal volume ventilation

Lung injury due to mechanical ventilation is associated with an impairment of endogenous surfactant. It is unknown whether this impairment is a consequence of or an active contributor to the development and progression of lung injury. To investigate this issue, the present study addressed three questions: Do alterations to surfactant precede physiological lung dysfunction during mechanical ventilation? Which components are responsible for surfactant's biophysical dysfunction? Does exogenous surfactant supplementation offer a physiological benefit in ventilation-induced lung injury? Adult rats were exposed to either a low-stretch [tidal volume (Vt) = 8 ml/kg, positive end-expiratory pressure (PEEP) = 5 cmH2O, respiratory rate (RR) = 54-56 breaths/min (bpm), fractional inspired oxygen (Fi(O2)) = 1.0] or high-stretch (Vt = 30 ml/kg, PEEP = 0 cmH2O, RR = 14-16 bpm, Fi(O2) = 1.0) ventilation strategy and monitored for either 1 or 2 h. Subsequently, animals were lavaged and the composition and function of surfactant was analyzed. Separate groups of animals received exogenous surfactant after 1 h of high-stretch ventilation and were monitored for an additional 2 h. High stretch induced a significant decrease in blood oxygenation after 2 h of ventilation. Alterations in surfactant pool sizes and activity were observed at 1 h of high-stretch ventilation and progressed over time. The functional impairment of surfactant appeared to be caused by alterations to the hydrophobic components of surfactant. Exogenous surfactant treatment after a period of high-stretch ventilation mitigated subsequent physiological lung dysfunction. Together, these results suggest that alterations of surfactant are a consequence of the ventilation strategy that impair the biophysical activity of this material and thereby contribute directly to lung dysfunction over time.     

Mechanical ventilation of isolated septic rat lungs: effects on surfactant and inflammatory cytokines.

The effects of mechanical ventilation (MV) on the surfactant system and cytokine secretion were studied in isolated septic rat lungs. At 23 h after sham surgery or induction of sepsis by cecal ligation and perforation (CLP), lungs were excised and randomized to one of three groups: 1) a nonventilated group, 2) a group subjected to 1 h of noninjurious MV (tidal volume = 10 ml/kg, positive end-expiratory pressure = 3 cmH(2)O), or 3) a group subjected to 1 h of injurious MV (tidal volume = 20 ml/kg, positive end-expiratory pressure = 0 cmH(2)O). Nonventilated sham and CLP lungs had similar compliance, normal lung morphology, surfactant, and cytokine concentrations. Injurious ventilation decreased compliance, altered surfactant, increased cytokines, and induced morphological changes compared with nonventilation in sham and CLP lungs. In these lungs, the surfactant system was similar in sham and CLP lungs; however, tumor necrosis factor-alpha and interleukin-6 levels were significantly higher in CLP lungs. We conclude that injurious ventilation altered surfactant independent of sepsis and that the CLP lungs were predisposed to the secretion of larger amounts of cytokines because of ventilation.     

Pulmonary mechanics during rapid mechanical ventilation in rabbits with saline-lavaged lungs

Infants with respiratory failure are frequently mechanically ventilated at rates exceeding 60 breaths/min. We analyzed the effect of ventilatory rates of 30, 60, and 90 breaths/min (inspiratory times of 0.6, 0.3, and 0.2 s, respectively) on the pressure-flow relationships of the lungs of anesthetized paralyzed rabbits after saline lavage. Tidal volume and functional residual capacity were maintained constant. We computed effective inspiratory and expiratory resistance and compliance of the lungs by dividing changes in transpulmonary pressure into resistive and elastic components with a multiple linear regression. We found that mean pulmonary resistance was lower at higher ventilatory rates, while pulmonary compliance was independent of ventilatory rate. The transpulmonary pressure developed by the ventilator during inspiration approximated a linear ramp. Gas flow became constant and the pressure-volume relationship linear during the last portion of inspiration. Even at a ventilatory rate of 90 breaths/min, 28-56% of the tidal volume was delivered with a constant inspiratory flow. Our findings are consistent with the model of Bates et al. (J. Appl. Physiol. 58: 1840-1848, 1985), wherein the distribution of gas flow within the lungs depends predominantly on resistive factors while inspiratory flow is increasing, and on elastic factors while inspiratory flow is constant. This dynamic behavior of the surfactant-depleted lungs suggests that, even with very short inspiratory times, distribution of gas flow within the lungs is in large part determined by elastic factors. Unless the inspiratory time is further shortened, gas flow may be directed to areas of increased resistance, resulting in hyperinflation and barotrauma.     

Serum SP-D is a marker of lung injury in rats

Pulmonary surfactant protein D (SP-D) is expressed in alveolar type II and bronchiolar epithelial cells and is secreted into alveoli and conducting airways. However, SP-D has also been measured in serum and is increased in patients with acute respiratory distress syndrome, pulmonary fibrosis, and alveolar proteinosis. To demonstrate that SP-D can be measured in rat serum, we instilled rats with keratinocyte growth factor, which produces type II cell hyperplasia and an increase in SP-D in bronchoalveolar lavage fluid (BALF). To evaluate serum SP-D as a biomarker of lung injury, we examined several injury models. In rats treated with 1 unit of bleomycin, serum SP-D was elevated on days 3, 7, 14, and 28 after instillation, and SP-D mRNA was increased in focal areas as detected by in situ hybridization. However, there was no increase in whole lung SP-D mRNA when the expression was normalized to whole lung 18S rRNA. After instillation of 2 units of bleomycin, the serum levels of SP-D were higher, and SP-D was also increased in BALF and lung homogenates. In another model of subacute injury, serum SP-D was increased in rats treated with paraquat plus oxygen. Finally to evaluate acute lung injury, we instilled rats with HCl; SP-D was increased at 4 h after instillation. Our data indicate that serum SP-D may be a useful indicator of lung injury and type II cell hyperplasia in rats.     

Early surfactant administration protects against lung dysfunction in a mouse model of ARDS

Sepsis can predispose the lung to insults such as mechanical ventilation (MV). It was hypothesized that treating the lung with exogenous surfactant early in the development of sepsis will reduce the lung dysfunction associated with MV 18 h later. Mice underwent sham or cecal ligation and perforation (CLP) surgery. Immediately after surgery, mice were either untreated or given 100 mg/kg of bovine lipid extract surfactant intratracheally. Eighteen hours later, the lungs were removed and analyzed either immediately or following ventilation ex vivo for 2 h by an "injurious" mode of ventilation (20 ml/kg, 0 cm positive end-expiratory pressure). In nonventilated lungs, exogenous surfactant had no impact on compliance or IL-6 concentrations in the lungs. In the ventilated groups, the administered surfactant had a significant protective effect on the lung dysfunction induced by MV, but only in the CLP lungs. We conclude that administration of exogenous surfactant at the time of a systemic insult can protect the lung from the damaging effects of MV 18 h later.     

Reverse right ventricular structural and extracellular matrix remodeling by estrogen in severe pulmonary hypertension

Acute respiratory distress syndrome is a pulmonary disease with a mortality rate of ～40% and 75,000 deaths annually in the United States. Mechanical ventilation restores airway patency and gas transport but leads to ventilator-induced lung injury. Furthermore, surfactant replacement therapy is ineffective due to surfactant delivery difficulties and deactivation by vascular proteins leaking into the airspace. Here, we demonstrated that surfactant function can be substantially improved (up to 50%) in situ in an in vitro pulmonary airway model using unconventional flows that incorporate a short-term retraction of the air-liquid interface, leading to a net decrease in cellular damage. Computational fluid dynamic simulations provided insights into this method and demonstrated the physicochemical hydrodynamic foundation for the improved surfactant microscale transport and mobility. This study may provide a starting point for developing novel ventilation waveforms to improve surfactant function in edematous airways.     

Lung protease/anti-protease network and modulation of mucus production and surfactant activity

Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local ‘alarm’ anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs.     

Metabolism of C-arachidonate in rat isolated lung

Metabolism of ¹⁴C-arachidonate was investigated in rat isolated lungs perfused via the pulmonary circulation with Krebs solution. Only 10% of the radioactivity derived from an infusion of ¹⁴C-arachidonate through the pulmonary circulation of rat isolated lungs appeared in the effluent by 10 minutes. At 10 min, the major component of effluent radioactivity and 20–40% of that retained in lung was unchanged arachidonate. Between 10 and 20 min of perfusion, a further small amount of radioactivity was lost in lung effluent and at 20 min the retained radioactivity showed a decrease in the proportion present as free arachidonate. Between 20 and 60 min, there was no further loss of radioactivity in effluent and no further change in the distribution in lung. Addition of albumin to the Krebs solution perfusate during the infusion of ¹⁴C-arachidonate increased effluent radioactivity to 80%, but albumin added after 10 min only caused the efflux of a small amount of radioactivity (10%). Treatment of labelled lung at 20 min with the calcium ionophore A23187 released biologically active metabolites of arachidonate but very little radioactivity. Metabolism of arachidonate, either during the infusion or after retention in lung, in rat lung was closer to that in human lung than to that in guinea-pig lung.     

Diffusivity dependence of multiple-breath washouts of lung periphery

Subpleural concentrations of He and SF6 were measured during multiple-breath washouts from isolated dog lungs. Tidal volume, inspiratory flow, and frequency were in the normal range of canine ventilation. For each gas, there was a local minimum in concentration during inspiration (Cinsp) and a local maximum in concentration during exhalation (Cexp). SF6 exhibited a deeper inspiratory trough than He for each breath of every washout. For large tidal volumes (10-20 ml/kg), Cexp approximated a single exponential decay and He was cleared more rapidly than SF6. For small tidal volumes (2.5 ml/kg), Cexp was multiexponential and SF6 was cleared more rapidly than He. Cinsp/Cexp (a measure of the depth of the inspiratory trough) and the kinetics of Cexp decay were determined for washouts using a tidal volume of 10 and 20 ml/kg and different inspiratory flows. Under all conditions, an increase of inspiratory flow resulted in a deeper inspiratory trough for both He and SF6. For washouts using 10 ml/kg and 60 breaths/min, an increase of inspiratory flow increased the clearance of both gases. In washouts using lower ventilatory frequencies, gas clearance was independent of inspiratory flow. These findings are contrary to predictions of contemporary models of convection and diffusion in the lung. This study suggests that convective axial mixing and radial diffusion in the airways are important determinants of pulmonary gas transport.