Lengths and topology of alveolar septal borders

To clarify the mechanics of alveolar parenchyma, we undertook a stereological and topological study in perfusion-fixed canine lungs of the borders of alveolar septa. We defined the principal borders as those along which one septum 1) joins two others (J), 2) joins one other at a distinct angle (B), or 3) joins no other structure (E). E and B borders are invariably reinforced with heavy connective tissue cables; J borders are not. Relative net lengths, determined from the number of traces per section area, were J, 45%; E, 19%; and B, 25%. These were remarkably constant over 10 canine lobes (5 animals, 4 volumes). Parenchyma, then, departs from the simple models that comprise only Js and Es. Bs are important; their net length exceeds that of Es. With lobe deflation, E shortened somewhat more than required to maintain geometric similarity, suggesting that the alveolar duct contracted disproportionately. A three-dimensional reconstruction was made from serial sections, and individual border segments were followed through the reconstruction. Typical lengths of individual J, B, and E borders were nearly equal. To characterize how the network of borders were interconnected, we counted the nodes at which they meet by class, e.g., EBE for the meeting of one B, two Es. The most common are JJJJ, 26%; EEEJ, 10%; EBJ, 24%; EBE, 8%; BBJJ, 12%. If parenchyma were constructed only from free-standing entrance rings and septal junctions, only JJJJ and EEEJ would be anticipated. The presence of EBJ, EBE, and BBJJ underscores parenchymal complexity. Only 7% of septa examined were bordered entirely by Js. Connective tissue cables were not confined to the alveolar duct's lumen but often extended to the primary septa at the periphery of the ductal unit. They rarely linked adjacent alveolar ducts; only 1 in 200 cable segments crossed from one duct to another. These observations support the concept that the parenchyma is an elastic network, characterized in part by a serial mechanical linkage from connective tissue cable to septal membrane to cable again.     

Respiratory mechanics and lung development in the rat from early age to adulthood.

The purpose of the present study was to establish how the dependence of respiratory mechanics on lung inflation changes during development. We studied seven groups of rats from 10 days to 3 mo of age at five levels of positive end-expiratory pressure (PEEP) from 0 to 7 hPa (1 hPa = 0.1 kPa approximately 1 cmH(2)O). At each PEEP level, we measured respiratory system resistance and elastance at both 0.9 and 4.8 Hz to partition the mechanical properties into its airway and tissue components. Elastance increased more rapidly with PEEP in the younger animals, which we interpret as reflecting a more pronounced strain stiffening of the younger parenchyma. However, the decrease in airway resistance with PEEP was more pronounced in the older animals. Morphometric analysis showed that mean tissue density decreased and total alveolar surface area increased with age. Our data suggest that the mechanical interdependence between airways and parenchyma is weaker in very young animals compared with mature animals. This may play a role in the hyperresponsiveness of immaturity.     

Continuum vs. spring network models of airway-parenchymal interdependence

The outward tethering forces exerted by the lung parenchyma on the airways embedded within it are potent modulators of the ability of the airway smooth muscle to shorten. Much of our understanding of these tethering forces is based on treating the parenchyma as an elastic continuum; yet, on a small enough scale, the lung parenchyma in two dimensions would seem to be more appropriately described as a discrete spring network. We therefore compared how the forces and displacements in the parenchyma surrounding a contracting airway are predicted to differ depending on whether the parenchyma is modeled as an elastic continuum or as a spring network. When the springs were arranged hexagonally to represent alveolar walls, the predicted parenchymal stresses and displacements propagated substantially farther away from the airway than when the springs were arranged in a triangular pattern or when the parenchyma was modeled as a continuum. Thus, to the extent that the parenchyma in vivo behaves as a hexagonal spring network, our results suggest that the range of interdependence forces due to airway contraction may have a greater influence than was previously thought.     

Atomic force microscope elastography reveals phenotypic differences in alveolar cell stiffness.

To understand the connection between alveolar mechanics and key biochemical events such as surfactant secretion, one first needs to characterize the underlying mechanical properties of the lung parenchyma and its cellular constituents. In this study, the mechanics of three major cell types from the neonatal rat lung were studied; primary alveolar type I (AT1) and type II (AT2) epithelial cells and lung fibroblasts were isolated using enzymatic digestion. Atomic force microscopy indentation was used to map the three-dimensional distribution of apparent depth-dependent pointwise elastic modulus. Histograms of apparent modulus data from all three cell types indicated non-Gaussian distributions that were highly skewed and appeared multimodal for AT2 cells and fibroblasts. Nuclear stiffness in all three cell types was similar (2.5+/-1.0 kPa in AT1 vs. 3.1+/-1.5 kPa in AT2 vs. 3.3+/-0.8 kPa in fibroblasts; n=10 each), whereas cytoplasmic moduli were significantly higher in fibroblasts and AT2 cells (6.0+/-2.3 and 4.7+/-2.9 kPa vs. 2.5+/-1.2 kPa). In both epithelial cell types, actin was arranged in sparse clusters, whereas prominent actin stress fibers were observed in lung fibroblasts. No systematic difference in actin or microtubule organization was noted between AT1 and AT2 cells. Atomic force microscope elastography, combined with live-cell fluorescence imaging, revealed that the stiffer measurements in AT2 cells often colocalized with lamellar bodies. These findings partially explain reported heterogeneity of alveolar cell deformation during in situ lung inflation and provide needed data for better understanding of how mechanical stretch influences surfactant release.     

Microscopic vs. macroscopic deformation of the pulmonary alveolar duct.

The stretch of the perimeters of alveolar ducts was measured at the surface of saline-filled specimens of human and dog lung parenchyma that were stretched biaxially. The microscopic stretch of these ducts was measured at several levels of isotropic biaxial macroscopic stretch of the parenchyma with stretch ratio (lambda x = lambda y) in the range of 1.20-1.40, which roughly corresponds to tidal breathing in humans and dogs. Alveolar walls were found to be load-carrying elements in the saline-filled lung, as seen by their straightness at all levels of stretch. Quantitatively, let l, A, L, and S denote, respectively, the duct perimeter length and area and the parenchymal target perimeter and area in the deformed state and lo, Ao, Lo, and So the corresponding variables in the undeformed state. The microscopic stretch ratio of the ducts (l/lo) was found to be approximately 4% larger than the macroscopic stretch ratio (L/Lo) in human lung and approximately 10% larger in dog lung. The microscopic area ratio of the ducts (A/Ao) was found to be approximately 10% larger than the macroscopic area ratio (S/So) in human lung and approximately 22% larger in dog lung. Ducts within human parenchyma were seen to be about twice as stiff as ducts within dog parenchyma over the range of macroscopic stretch studied. This correlates with the volume fractions of collagen and elastin being higher in the human lung than in dog lung. The observed nonuniformity in strain field at the microstructural level suggests the need to include a force balance between alveolar ducts and septal walls when modeling the mechanics of saline-filled parenchyma.     

Mechanics of edematous lungs.

Using the parenchymal marker technique, we measured pressure (P)-volume (P-V) curves of regions with volumes of approximately 1 cm3 in the dependent caudal lobes of oleic acid-injured dog lungs, during a very slow inflation from P = 0 to P = 30 cmH2O. The regional P-V curves are strongly sigmoidal. Regional volume, as a fraction of volume at total lung capacity, remains constant at 0.4-0.5 for airway P values from 0 to approximately 20 cmH2O and then increases rapidly, but continuously, to 1 at P = approximately 25 cmH2O. A model of parenchymal mechanics was modified to include the effects of elevated surface tension and fluid in the alveolar spaces. P-V curves calculated from the model are similar to the measured P-V curves. At lower lung volumes, P increases rapidly with lung volume as the air-fluid interface penetrates the mouth of the alveolus. At a value of P = approximately 20 cmH2O, the air-fluid interface is inside the alveolus and the lung is compliant, like an air-filled lung with constant surface tension. We conclude that the properties of the P-V curve of edematous lungs, particularly the knee in the P-V curve, are the result of the mechanics of parenchyma with constant surface tension and partially fluid-filled alveoli, not the result of abrupt opening of airways or atelectatic parenchyma.     

Alveolar pressure measurement in open-chest rats.

In open-chest rats, alveolar pressure was measured with alveolar capsules connected via pliable tubing to inductive pressure transducers. By means of the interrupter technique during constant-flow inflation, it was possible to determine pulmonary static elastance (Est,L) and tissue and airway resistances (Rdiff,L and Rinit,L, respectively). In eight anesthetized paralyzed mechanically ventilated rats, 118 measurements of Rdiff,L and Est,L were performed over a wide range of flows and tidal volumes. There was excellent agreement between the data calculated using transpulmonary pressures and those computed using capsule pressures, the latter being measured at different points of the lung. In another group of rats studied under the same experimental conditions, two capsules were simultaneously placed on different pulmonary lobes. No regional differences in pulmonary mechanics could be detected in either experiment. In addition, alveolar pressure could also be measured accurately by a catheter inserted into lung parenchyma.     

Pneumonitis and emphysema in sp-C gene targeted mice

SP-C-deficient (SP-C -/-) mice developed a severe pulmonary disorder associated with emphysema, monocytic infiltrates, epithelial cell dysplasia, and atypical accumulations of intracellular lipids in type II epithelial cells and alveolar macrophages. Whereas alveolar and tissue surfactant phospholipid pools were increased, levels of other surfactant proteins were not altered (SP-B) or were modestly increased (SP-A and SP-D). Analysis of pressure-volume curves and forced oscillatory dynamics demonstrated abnormal respiratory mechanics typical of emphysema. Lung disease was progressive, causing weight loss and cardiomegaly. Extensive alveolar remodeling was accompanied by type II cell hyperplasia, obliteration of pulmonary capillaries, and widespread expression of alpha-smooth muscle actin, indicating myofibroblast transformation in the lung parenchyma. Dysplastic epithelial cells lining conducting airways stained intensely for the mucin, MUC5A/C. Tissue concentrations of proinflammatory cytokines were not substantially altered in the SP-C (-/-) mice. Production of matrix metalloproteinases (MMP-2 and MMP-9) was increased in alveolar macrophages from SP-C (-/-) mice. Absence of SP-C caused a severe progressive pulmonary disorder with histologic features consistent with interstitial pneumonitis.     

What do we know about mechanical strain in lung alveoli?

The pulmonary alveolus, terminal gas-exchange unit of the lung, is composed of alveolar epithelial and endothelial cells separated by a thin basement membrane and interstitial space. These cells participate in the maintenance of a delicate system regulated not only by biological factors but also by the mechanical environment of the lung, which undergoes dynamic deformation during breathing. Clinical and animal studies as well as cell culture studies point toward a strong influence of mechanical forces on lung cells and tissues including effects on growth and repair, surfactant release, injury, and inflammation. However, despite substantial advances in our understanding of lung mechanics over the last half century, there are still many unanswered questions regarding the micromechanics of the alveolus and how it deforms during lung inflation. Therefore, the aims of this review are to draw a multidisciplinary account of the mechanics of the alveolus on the basis of its structure, biology, and chemistry and to compare estimates of alveolar deformation from previous studies.     

Stereologic analysis of the respiratory zone of the lungs of the laboratory rat and man during aging

By means of stereological analysis methods, lungs of mature and ageing laboratory rats and those of human beings at the age of 21-40, 41-60 and further have been studied. During the process of ageing the fraction of nonparenchymatous structures increases, while that of parenchyma decreases. Architectonics of acini is described: the volume of central passages increases and the volume of alveolar air decreases. The form of the alveoli approaches the spherical one. The area of the internal surface and the alveolar volume increase. The total number of the alveoli and the area of the respiratory lining decrease. The total number of the alveolar capillaries does not change, however, the density of their distribution grows small. The area and volume of a separate segment of the alveolar capillaries grow large. The age rearrangement of the respiratory zone morphologically differs from the changes that are observed at the obstructive emphysema.     

Partitioning of pulmonary responses to inhaled methacholine in puppies.

Twelve open-chest mongrel puppies, 8-10 wk old, were studied to localize the site of action of inhaled methacholine within the lungs. Six puppies were challenged with methacholine aerosols and six were challenged with an equal number of nebulizations of normal saline (control group). Pulmonary mechanics were measured during mechanical ventilation and after midexpiratory flow interruptions. Alveolar pressure was measured to allow the partitioning of pulmonary mechanics into airway and tissue components. Good matching between airway opening and alveolar pressures was seen throughout the study. After methacholine challenge, lung resistance increased fivefold. Increases in airway resistance and in the parameters reflecting tissue viscoelastic properties contributed to this increase in lung resistance. Dynamic lung elastance also increased threefold. The response of the methacholine group was statistically different from that of the control group. These data indicate that both the airways and pulmonary parenchyma contribute to the response to inhaled methacholine in 8- to 10-wk-old puppies.     

Vitamin A deficiency alters the pulmonary parenchymal elastic modulus and elastic fiber concentration in rats

Background

Bronchial hyperreactivity is influenced by properties of the conducting airways and the surrounding pulmonary parenchyma, which is tethered to the conducting airways. Vitamin A deficiency (VAD) is associated with an increase in airway hyperreactivity in rats and a decrease in the volume density of alveoli and alveolar ducts. To better define the effects of VAD on the mechanical properties of the pulmonary parenchyma, we have studied the elastic modulus, elastic fibers and elastin gene-expression in rats with VAD, which were supplemented with retinoic acid (RA) or remained unsupplemented.

Methods

Parenchymal mechanics were assessed before and after the administration of carbamylcholine (CCh) by determining the bulk and shear moduli of lungs that that had been removed from rats which were vitamin A deficient or received a control diet. Elastin mRNA and insoluble elastin were quantified and elastic fibers were enumerated using morphometric methods. Additional morphometric studies were performed to assess airway contraction and alveolar distortion.

Results

VAD produced an approximately 2-fold augmentation in the CCh-mediated increase of the bulk modulus and a significant dampening of the increase in shear modulus after CCh, compared to vitamin A sufficient (VAS) rats. RA-supplementation for up to 21 days did not reverse the effects of VAD on the elastic modulus. VAD was also associated with a decrease in the concentration of parenchymal elastic fibers, which was restored and was accompanied by an increase in tropoelastin mRNA after 12 days of RA-treatment. Lung elastin, which was resistant to 0.1 N NaOH at 98°, decreased in VAD and was not restored after 21 days of RA-treatment.

Conclusion

Alterations in parenchymal mechanics and structure contribute to bronchial hyperreactivity in VAD but they are not reversed by RA-treatment, in contrast to the VAD-related alterations in the airways.     

Immunohistochemical detection of nicotinic acetylcholine receptor subunits alpha9 and alpha10 in rat lung isografts and allografts

The success of clinical lung transplantation is poor in comparison to other solid organ transplants and novel therapeutic approaches are badly needed. In the view of the recent discovery of anti-inflammatory pathways mediated via nicotinic acetylcholine receptors, we investigated changes in this system in pulmonary isografts and allografts by immunohistochemistry. Lung transplantation was performed in the isogeneic Lewis to Lewis rat strain combination. For allogeneic transplantation Dark Agouti rats were used as donors. Nicotinic alpha9 and alpha10 acetylcholine receptor subunits were detected on alveolar macrophages as well as in the lung parenchyma of native and transplanted lungs. The expression of both receptor subunits was up-regulated in the parenchyma of day 4 allografts. These allografts were characterized by accumulations of alveolar macrophages strongly expressing the alpha9 and the alpha10 receptor subunit. Therapeutic application of nicotinic agonists might down-modulate pro-inflammatory functions of alveolar macrophages and protect pulmonary transplants.     

Ventilation inhomogeneity: Alveolar mechanics and gas distribution

The effects of regional lung differences in alveolar mechanics on the transpulmonary pressure-volume (P_tp-V) relationship and the single-breath washout (SBW) of nitrogen were investigated by mathematical modeling and postmorten human lung experiments. Regional nonuniformity in alveolar collapse and re-opening were associated with differences in gravitational stress or elasticity. Model simulations predict that neither type of regional nonuniformity qualitatively affects the shape of the P_tp-V curve, but does affect the terminal (or small-volume) portion of the SBW. Comparisons of characteristics of the P_tp-V and SBW curves indicate that regional nonuniformity in alveolar collapse is an important mechanism associated with ventilation inhomogeneity.     

A simple method for the differential characterization of alveoli and alveolar ducts in injured lungs

RATIONALE AND HYPOTHESIS: Previous studies evaluating the histoarchitecture of distal airspaces have been shown to be limited by the difficulty in adequately differentiating alveoli and alveolar ducts. This limitation has been specially noticed in studies addressing lung recruitment and in situations of diffuse alveolar damage (DAD), where generic nominations for distal airspaces had to be created, such as "peripheral airspaces" (PAS) and "large-volume gas-exchanging airspaces" (LVGEA). Elastic stains have been largely used to describe normal lung structures. Weigert's resorcin-fuchsin staining (WRF) demarcates the thickened free portions of the ductal septum facilitating its recognition. We hypothesized that this staining could help in differentiating alveoli from alveolar ducts in distorted lung parenchyma. MATERIAL AND METHODS: Samples of control lungs and of DAD lungs induced by mechanical ventilation (VILI) were stained with hematoxylin-eosin (HE) and with WRF. Using morphometry we assessed the volume proportion of alveoli, alveolar ducts and LVGEA in control and VILI lungs. RESULTS: WRF stained VILI lungs showed a significant decrease in the volume proportion of LVGEA and alveoli and a significant increase in the volume proportion of alveolar ducts when compared to HE stained samples. CONCLUSION: We conclude that WRF staining is useful to distinguish alveolar ducts from alveoli in a DAD model, and suggest that it should be routinely used when morphometric studies of lung parenchyma are performed.     

Comparison of amounts of collagen and elastin in pleura and parenchyma of dog lung

Pressure-volume characteristics of the lung have been thought to be due primarily to the properties of the network of alveolar septa. However, Hajji et al. (J. Appl. Physiol.: Respirat . Environ. Exercise Physiol. 47: 175-181, 1979) attributed a substantial role to the visceral pleura. Seeking a structural explanation for this result, we compared the relative amounts of collagen fibrils and elastin fibers in the visceral pleura and alveolar parenchyma using stereological measurements in five canine lobes. We found about one-fifth as much collagen and one-tenth as much elastin in the pleura as in the alveolar parenchyma. This structural result confirms the functional conclusions of Hajji et al. We argue that such a substantial structure is not needed for protection against overinflation but may have to do with stabilization of lobe shape or handling of frictional forces.     

Comparison of early and late responses to antigen of sensitized guinea pig parenchymal lung strips.

The peripheral lung parenchyma has been studied as a component of the asthmatic inflammatory response. During induced constriction, tissue resistance increases in different asthma models. Approximately 60% of the asthmatic patients show early and late responses. The late response is characterized by more severe airway obstruction. In the present study, we evaluated lung parenchymal strips mechanics in ovalbumin-sensitized guinea pigs, trying to reproduce both early and late inflammatory responses. Oscillatory mechanics of lung strips were performed in a control group (C), in an early response group (ER), and in two late response groups: 17 h (L1) and 72 h (L2) after the last ovalbumin challenge. Measurements of resistance and elastance were obtained before and after ovalbumin challenge in C and ER groups and before and after acetylcholine challenge in all groups. Using morphometry, we assessed the density of eosinophils and smooth muscle cells, as well as collagen and elastin content in lung strips. The baseline and postagonist values of resistance and elastance were increased in ER, L1, and L2 groups compared with C (P < or = 0.001). The morphometric analysis showed an increase in alveolar eosinophil density in ER and L2 groups compared with C (P < 0.05). There was a significant correlation between eosinophil density in parenchymal strips of C, L1, and L2 groups and values of resistance and elastance postacetylcholine (r = 0.71, P = 0.001 and r = 0.74, P < 0.001, respectively). The results show that the lung parenchyma is involved in the late response, and the constriction response in this phase is related to the eosinophilic inflammation.     

Depletion of pulmonary EC-SOD after exposure to hyperoxia

Extracellular superoxide dismutase (EC-SOD) is highly expressed in lung tissue. EC-SOD contains a heparin-binding domain that is sensitive to proteolysis. This heparin-binding domain is important in allowing EC-SOD to exist in relatively high concentrations in specific regions of the extracellular matrix and on cell surfaces. EC-SOD has been shown to protect the lung against hyperoxia in transgenic and knockout studies. This study tests the hypothesis that proteolytic clearance of EC-SOD from the lung during hyperoxia contributes to the oxidant-antioxidant imbalance that is associated with this injury. Exposure to 100% oxygen for 72 h resulted in a significant decrease in EC-SOD levels in the lungs and bronchoalveolar lavage fluid of mice. This correlated with a significant depletion of EC-SOD from the alveolar parenchyma as determined by immunofluorescence and immunohistochemistry. EC-SOD mRNA was unaffected by hyperoxia; however, there was an increase in the ratio of proteolyzed to uncut EC-SOD after hyperoxia, which suggests that hyperoxia depletes EC-SOD from the alveolar parenchyma by cutting the heparin-binding domain. This may enhance hyperoxic pulmonary injury by altering the oxidant-antioxidant balance in alveolar spaces.     

Contribution of alveolar macrophages to the response of the TIMP-3 null lung during a septic insult

Mice deficient in tissue inhibitor of metalloproteinase-3 (TIMP-3) develop an emphysema-like phenotype involving increased pulmonary compliance, tissue degradation, and matrix metalloproteinase (MMP) activity. After a septic insult, they develop a further increase in compliance that is thought to be a result of heightened metalloproteinase activity produced by the alveolar macrophage, potentially modeling an emphysemic exacerbation. Therefore, we hypothesized that TIMP-3 null mice lacking alveolar macrophages would not be susceptible to the altered lung function associated with a septic insult. TIMP-3 null and wild-type (WT) mice were depleted of alveolar macrophages before the induction of a septic insult and assessed for alteration in lung mechanics, alveolar structure, metalloproteinase levels, and inflammation. The results showed that TIMP-3 null mice lacking alveolar macrophages were protected from sepsis-induced alterations in lung mechanics, particularly pulmonary compliance, a finding that was supported by changes in alveolar structure. Additionally, changes in lung mechanics involved primarily peripheral tissue vs. central airways as determined using the flexiVent system. From investigation into possible molecules that could cause these alterations, it was found that although several proteases and inflammatory mediators were increased during the septic response, only MMP-7 was attenuated after macrophage depletion. In conclusion, the alveolar macrophage is essential for the TIMP-3 null sepsis-induced compliance alterations. This response may be mediated in part by MMP-7 activity but occurs independently of inflammatory cytokine and/or chemokine concentrations.     

Morphometric analyses of the effects of thyrotrophin releasing hormone and cortisol on the lungs of fetal sheep

Morphometric analyses of ovine fetal lung parenchyma were undertaken in order to elucidate the roles of pituitary, thyroid and adrenocortical hormones in promoting the structural changes underlying the increased distensibility and stability present in mature fetal lungs. Twenty-six Romney fetuses were treated with either cortisol for 84 h from 125 days (4), pulsatile TRH for 6.5 days from 122 days (4), cortisol and TRH (12), or 0.9% NaCl solution (6). The left lungs were used for physiological studies (distensibility, V40) and the right lungs were prepared for electron microscopy. Using 32 regions of lung parenchyma per fetus, volume density, surface density and arithmetic mean thickness of the alveolar walls were calculated using point and intersection counts. Of the three regimens, treatment with TRH + cortisol (exposure to raised concentrations of cortisol, T3 and prolactin) induced significantly greater lung distensibility, the largest potential alveolar air space (62% of the parenchyma), the greatest alveolar surface area (113.7 mm2/mm3 x 10(-3)) and the thinnest alveolar walls (6.7 microns). We conclude that cortisol, T3 and prolactin act synergistically to promote maturational changes in the alveolar wall. While cortisol plays the major role, T3 and prolactin enhance the ability of the immature lung to respond to the cortisol.