Multiscale stiffness of human emphysematous precision cut lung slices.

Emphysema is a debilitating disease that remodels the lung leading to reduced tissue stiffness. Thus, understanding emphysema progression requires assessing lung stiffness at both the tissue and alveolar scales. Here, we introduce an approach to determine multiscale tissue stiffness and apply it to precision-cut lung slices (PCLS). First, we established a framework for measuring stiffness of thin, disk-like samples. We then designed a device to verify this concept and validated its measuring capabilities using known samples. Next, we compared healthy and emphysematous human PCLS and found that the latter was 50% softer. Through computational network modeling, we discovered that this reduced macroscopic tissue stiffness was due to both microscopic septal wall remodeling and structural deterioration. Lastly, through protein expression profiling, we identified a wide spectrum of enzymes that can drive septal wall remodeling, which, together with mechanical forces, lead to rupture and structural deterioration of the emphysematous lung parenchyma.

Predicting alveolar ventilation heterogeneity in pulmonary fibrosis using a non-uniform polyhedral spring network model.

Pulmonary Fibrosis (PF) is a deadly disease that has limited treatment options and is caused by excessive deposition and cross-linking of collagen leading to stiffening of the lung parenchyma. The link between lung structure and function in PF remains poorly understood, although its spatially heterogeneous nature has important implications for alveolar ventilation. Computational models of lung parenchyma utilize uniform arrays of space-filling shapes to represent individual alveoli, but have inherent anisotropy, whereas actual lung tissue is isotropic on average. We developed a novel Voronoi-based 3D spring network model of the lung parenchyma, the Amorphous Network, that exhibits more 2D and 3D similarity to lung geometry than regular polyhedral networks. In contrast to regular networks that show anisotropic force transmission, the structural randomness in the Amorphous Network dissipates this anisotropy with important implications for mechanotransduction. We then added agents to the network that were allowed to carry out a random walk to mimic the migratory behavior of fibroblasts. To model progressive fibrosis, agents were moved around the network and increased the stiffness of springs along their path. Agents migrated at various path lengths until a certain percentage of the network was stiffened. Alveolar ventilation heterogeneity increased with both percent of the network stiffened, and walk length of the agents, until the percolation threshold was reached. The bulk modulus of the network also increased with both percent of network stiffened and path length. This model thus represents a step forward in the creation of physiologically accurate computational models of lung tissue disease.

Remodeling of the Aged and Emphysematous Lungs: Roles of Microenvironmental Cues.

Aging is a slow process that affects all organs, and the lung is no exception. At the alveolar level, aging increases the airspace size with thicker and stiffer septal walls and straighter and thickened collagen and elastic fibers. This creates a microenvironment that interferes with the ability of cells in the parenchyma to maintain normal homeostasis and respond to injury. These changes also make the lung more susceptible to disease such as emphysema. Emphysema is characterized by slow but progressive remodeling of the deep alveolar regions that leads to airspace enlargement and increased but disorganized elastin and collagen deposition. This remodeling has been attributed to ongoing inflammation that involves inflammatory cells and the cytokines they produce. Cellular senescence, another consequence of aging, weakens the ability of cells to properly respond to injury, something that also occurs in emphysema. These factors conspire to make alveolar walls more prone to mechanical failure, which can set emphysema in motion by driving inflammation through immune stimulation by protein fragments. Both aging and emphysema are influenced by microenvironmental conditions such as local inflammation, chemical makeup, tissue stiffness, and mechanical stresses. Although aging and emphysema are not equivalent, they have the potential to influence each other in synergistic ways; aging sets up the conditions for emphysema to develop, while emphysema may accelerate cellular senescence and thus aging itself. This article focuses on the similarities and differences between the remodeled microenvironment of the aging and emphysematous lung, with special emphasis on the alveolar septal wall. © 2022 American Physiological Society. Compr Physiol 12:3559-3574, 2022.

Mechano-inflammatory sensitivity of ACE2: Implications for the regional distribution of SARS-CoV-2 injury in the lung.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 can result in severe injury to the lung. Computed tomography images have revealed that the virus preferentially affects the base of the lung, which experiences larger tidal stretches than the apex. We hypothesize that the expression of both the angiotensin converting enzyme-2 (ACE2) receptor for SARS-CoV-2 and the transmembrane serine protease 2 (TMPRSS2) are sensitive to regional cell stretch in the lung. To test this hypothesis, we stretched precision cut lung slices (PCLS) for 12 h with one of the following protocols: 1) unstretched (US); 2) low-stretch (LS), 5% peak-to-peak area strain mimicking the lung base; or 3) high-stretch (HS), the same peak-to-peak area strain superimposed on 10% static area stretch mimicking the lung apex. PCLS were additionally stretched in cigarette smoke extract (CSE) to mimic an acute inflammatory exposure. The expression of ACE2 was higher whereas that of TMPRSS2 was lower in the control samples following LS than HS. CSE-induced inflammation substantially altered the expression of ACE2 with higher levels following HS than LS. These results suggest that ACE2 and TMPRSS2 expression in lung cells is mechanosensitive, which could have implications for the spatial distribution of COVID-19-mediated lung injury and the increased risk for more severe disease in active smokers and patients with COPD.

Stabilizing breathing pattern using local mechanical vibrations: comparison of deterministic and stochastic stimulations in rodent models of apnea of prematurity.

Mechanical stimulation has been shown to reduce apnea of prematurity (AOP), a major concern in preterm infants. Previous work suggested that the underlying mechanism is stochastic resonance, amplification of a subthreshold signal by stochastic stimulation. We hypothesized that the mechanism behind the reduction of apnea length may not be a solely stochastic phenomenon, and suggest that a purely deterministic, non-random mechanical stimulation could be equally as effective. Mice and rats were anesthetized, tracheostomized, and mechanically ventilated to halt spontaneous breathing. Two miniature motors controlled by a microcontroller were attached around the abdomen. Ventilation was paused, stimulations were applied, and the time to the rodent's first spontaneous breath (T) was measured. Six spectrally different signals were compared to one another and the no-stimulation control in mice. The most successful deterministic stimulation (D) at reducing apnea was then compared to a pseudo-random noise (PRN) signal of comparable amplitude and frequency. CO2%, CO2 stabilization time (Ts), O2 saturation (SpO2%), and T were also measured. D significantly reduced T compared to no stimulation for medium and high amplitudes. PRN also reduced T, without  a difference between D and PRN. Furthermore, both stimulations significantly reduced Ts with no significant differences between the respective stimulations. However, there was no effect of D or PRN on SpO2%. The lack of differences between D and PRN led to an additional series of experiment comparing the same D to a band-limited white noise (WN) signal in young rats. Both D and WN were shown to significantly reduce T, with D showing statistical superiority in reduction of apnea. We further speculate that both deterministic and stochastic mechanical stimulations induce some form of mechanotransduction which is responsible for their efficacy, and our findings suggest that mechanical stimulation may be effective in treating AOP. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13534-021-00203-x.

Evaluation of 1-mm-diameter endothelialized dense collagen tubes in vascular microsurgery.

Although much progress has been made in engineering vascular grafts for large- and small-diameter arterial repair or bypass, the extension of these results to the microsurgical size scale has been challenging. Here, we evaluated the use of dense collagen tubes (outer diameter 1 mm, inner diameter 0.5 mm) for vascular microsurgery as interpositional grafts to the femoral artery of Lewis rats. These tubes were formed by dehydrating tubular collagen gels around a mandrel, crosslinking them with genipin, seeding with syngeneic endothelial cells, and culturing before implantation by suture anastomosis. The retention of a confluent endothelial lining inside the tubes after mock surgical handling depended strongly on the crosslinker concentration and culture time. Optimized preparation conditions enabled retention of endothelium after mock surgical handling in ~80% of tubes and maintenance of patency 7 days after implantation in ~40% of grafts. Histological analysis showed the development of granulation tissue and the presence of CD31-positive structures on the inner and outer surfaces of implants. This study provides a proof-of-principle demonstration that endothelialized dense collagen tubes can remain patent for up to 7 days after vascular microsurgery, and points to the importance of mild scaffold crosslinking for maintaining firm endothelial adhesion.

Tuning mitochondrial structure and function to criticality by fluctuation-driven mechanotransduction.

Cells in vascular walls are exposed to blood pressure variability (BPV)-induced cycle-by-cycle fluctuations in mechanical forces which vary considerably with pathology. For example, BPV is elevated in hypertension but reduced under anesthesia. We hypothesized that the extent of mechanical fluctuations applied to vascular smooth muscle cells (VSMCs) regulates mitochondrial network structure near the percolation transition, which also influences ATP and reactive oxygen species (ROS) production. We stretched VSMCs in culture with cycle-by-cycle variability in area strain ranging from no variability (0%), as in standard laboratory conditions, through abnormally small (6%) and physiological (25%) to pathologically high (50%) variability mimicking hypertension, superimposed on 0.1 mean area strain. To explore how oxidative stress and ATP-dependent metabolism affect mitochondria, experiments were repeated in the presence of hydrogen peroxide and AMP-PNP, an ATP analog and competitive inhibitor of ATPases. Physiological 25% variability maintained activated mitochondrial cluster structure at percolation with a power law distribution and exponent matching the theoretical value in 2 dimensions. The 25% variability also maximized ATP and minimized cellular and mitochondrial ROS production via selective control of fission and fusion proteins (mitofusins, OPA1 and DRP1) as well as through stretch-sensitive regulation of the ATP synthase and VDAC1, the channel that releases ATP into the cytosol. Furthermore, pathologically low or high variability moved mitochondria away from percolation which reduced the effectiveness of the electron transport chain by lowering ATP and increasing ROS productions. We conclude that normal BPV is required for maintaining optimal mitochondrial structure and function in VSMCs.

Blood pressure-induced physiological strain variability modulates wall structure and function in aorta rings.

Vascular smooth muscle cells respond to mechanical stretch by reorganizing their cytoskeletal and contractile elements. Recently, we showed that contractile forces in rat aorta rings were maintained when the rings were exposed to 4 h of physiological variability in cycle-by-cycle strain, called variable stretch (VS), mimicking beat-to-beat blood pressure variability. Contractility, however, was reduced when the aorta was exposed to monotonous stretch (MS) with an amplitude equal to the mean peak strain of VS. OBJECTIVE: Here we reanalyzed the data to obtain wall stiffness as well as added new histologic and inhibitor studies to test the effects of VS on the extracellular matrix. MAIN RESULTS: The results demonstrate that while the stiffness of the aorta did not change during 4 h MS or VS, nonlinearity in mechanical behavior was slightly stronger following MS. The inhibitor studies also showed that mitochondrial energy production and cytoskeletal organization were involved in this fluctuation-driven mechanotransduction. Reorganization of ß-actin in the smooth muscle layer quantified from immunohistochemically labeled images correlated with contractile forces during contraction. Histologic analysis of wall structure provided evidence of reorganization of elastin and collagen fibers following MS but less so following VS. The results suggested that the loss of muscle contraction in MS was compensated by reorganization of fiber structure leading to similar wall stiffness as in VS. SIGNIFICANCE: We conclude that muscle tone modulated by variability in stretch plays a role in maintaining aortic wall structural and mechanical homeostasis with implications for vascular conditions characterized by a loss or an increase in blood pressure variability.

The effect of mechanical or electrical stimulation on apnea length in mice.

Premature birth is a leading cause of infant mortality which is often attributed to irregular breathing and apnea of prematurity. A common treatment for apnea is caffeine to stimulate the brain's respiratory center. However, caffeine's long term effect on infant development is not fully comprehended. We hypothesized that noninvasive localized body stimulation regularizes breathing pattern. We investigated the impact of electrical or mechanical stimulation on breathing in mice. After the mice were ventilated for 28 s to induce apnea, mice were taken off the ventilator while receiving mechanical, electrical, or no stimulation in a randomized order. Both stimuli targeted the diaphragm area through a custom-built belt with vibrating motors or adhesive electrodes. After each apnea cycle, the time to take the first breath (T) was recorded. The electrical stimulation given at 4.5, 8.3, 16.7 V (pulse rate = 3 Hz, pulse width = 120 µs) showed no reduction in T. Electrical stimulation at pulse rates of 10 or 20 Hz (16.7 V, pulse width 260 µs) showed a detrimental effect increasing T by ~ 7% compared to control values (p = 0.005, p = 0.038 respectively). High and medium intensity mechanical stimulations significantly reduced T by 11.74 (p < 10-13) and by 17.08% (p < 10-8), respectively. Further reducing the amplitude of vibrations did not affect T. When the probe was attached to the ankles, only the high intensity vibrations resulted in a decrease in T (p < 10-13). Mechanical vibrations, applied at various intensities and locations, could be used to treat irregular breathing and apnea in infants.

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors.

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging.

Elastase-Induced Lung Emphysema Models in Mice.

Pulmonary emphysema is one of the distinct pathological forms of chronic obstructive pulmonary disease (COPD) that is accompanied by gradual elimination of alveolar tissue, causing reductions in lung recoil and leading to difficulty in breathing. As there is no cure for emphysema, animal models are often used to better understand the pathogenesis and progression of the disease. One widely used animal model of emphysema is the elastase treatment. In this chapter, we describe two methods of elastase-induced emphysema in mice. The first is a single-dose treatment, whereby elastase is introduced oropharengeally into the lung and the structure and/or function of the lungs are studied between 2 days and 4 weeks following the treatment. The second method consists of exposing mice repeatedly (four times) to elastase intratracheally and observing the effects of the treatment 1-4 weeks following the last administration of the enzyme. Both protocols are described in detail, and examples of lung structure and function of the emphysematous mouse lung are provided.

Optimization of Variable Ventilation for Physiology, Immune Response and Surfactant Enhancement in Preterm Lambs.

Preterm infants often require mechanical ventilation due to lung immaturity including reduced or abnormal surfactant. Since cyclic stretch with cycle-by-cycle variability is known to augment surfactant release by epithelial cells, we hypothesized that such in vivo mechanotransduction improves surfactant maturation and hence lung physiology in preterm subjects. We thus tested whether breath-by-breath variability in tidal volume (VT) in variable ventilation (VV) can be tuned for optimal performance in a preterm lamb model. Preterm lambs were ventilated for 3 h with conventional ventilation (CV) or two variants of VV that used a maximum VT of 1.5 (VV1) or 2.25 (VV2) times the mean VT. VT was adjusted during ventilation to a permissive pCO2 target range. Respiratory mechanics were monitored continuously using the forced oscillation technique, followed by postmortem bronchoalveolar lavage and tissue collection. Both VVs outperformed CV in blood gas parameters (pH, SaO2, cerebral O2 saturation). However, only VV2 lowered PaCO2 and had a higher specific respiratory compliance than CV. VV2 also increased surfactant protein (SP)-B release compared to VV1 and stimulated its production compared to CV. The production and release of proSP-C however, was increased with CV compared to both VVs. There was more SP-A in both VVs than CV in the lung, but VV2 downregulated SP-A in the lavage, whereas SP-D significantly increased in CV in both the lavage and lung. Compared to CV, the cytokines IL-1ß, and TNFα decreased with both VVs with less inflammation during VV2. Additionally, VV2 lungs showed the most homogeneous alveolar structure and least inflammatory cell infiltration assessed by histology. CV lungs exhibited over-distension mixed with collapsed and interstitial edematous regions with occasional hemorrhage. Following VV1, some lambs had normal alveolar structure while others were similar to CV. The IgG serum proteins in the lavage, a marker of leakage, were the highest in CV. An overall combined index of performance that included physiological, biochemical and histological markers was the best in VV2 followed by VV1. Thus, VV2 outperformed VV1 by enhancing SP-B metabolism resulting in open alveolar airspaces, less leakage and inflammation and hence better respiratory mechanics.

A microfluidic chamber-based approach to map the shear moduli of vascular cells and other soft materials.

There is growing interest in quantifying vascular cell and tissue stiffness. Most measurement approaches, however, are incapable of assessing stiffness in the presence of physiological flows. We developed a microfluidic approach which allows measurement of shear modulus (G) during flow. The design included a chamber with glass windows allowing imaging with upright or inverted microscopes. Flow was controlled gravitationally to push culture media through the chamber. Fluorescent beads were conjugated to the sample surface and imaged before and during flow. Bead displacements were calculated from images and G was computed as the ratio of imposed shear stress to measured shear strain. Fluid-structure simulations showed that shear stress on the surface did not depend on sample stiffness. Our approach was verified by measuring the moduli of polyacrylamide gels of known stiffness. In human pulmonary microvascular endothelial cells, G was 20.4 ± 12 Pa and decreased by 20% and 22% with increasing shear stress and inhibition of non-muscle myosin II motors, respectively. The G showed a larger intra- than inter-cellular variability and it was mostly determined by the cytosol. Our shear modulus microscopy can thus map the spatial distribution of G of soft materials including gels, cells and tissues while allowing the visualization of microscopic structures such as the cytoskeleleton.

Regulatory Roles of Fluctuation-Driven Mechanotransduction in Cell Function.

Cells in the body are exposed to irregular mechanical stimuli. Here, we review the so-called fluctuation-driven mechanotransduction in which stresses stretching cells vary on a cycle-by-cycle basis. We argue that such mechanotransduction is an emergent network phenomenon and offer several potential mechanisms of how it regulates cell function. Several examples from the vasculature, the lung, and tissue engineering are discussed. We conclude with a list of important open questions.

Mechanical Forces Accelerate Collagen Digestion by Bacterial Collagenase in Lung Tissue Strips.

Most tissues in the body are under mechanical tension, and while enzymes mediate many cellular and extracellular processes, the effects of mechanical forces on enzyme reactions in the native extracellular matrix (ECM) are not fully understood. We hypothesized that physiological levels of mechanical forces are capable of modifying the activity of collagenase, a key remodeling enzyme of the ECM. To test this, lung tissue Young's modulus and a nonlinearity index characterizing the shape of the stress-strain curve were measured in the presence of bacterial collagenase under static uniaxial strain of 0, 20, 40, and 80%, as well as during cyclic mechanical loading with strain amplitudes of ±10 or ±20% superimposed on 40% static strain, and frequencies of 0.1 or 1 Hz. Confocal and electron microscopy was used to determine and quantify changes in ECM structure. Generally, mechanical loading increased the effects of enzyme activity characterized by an irreversible decline in stiffness and tissue deterioration seen on both confocal and electron microscopic images. However, a static strain of 20% provided protection against digestion compared to both higher and lower strains. The decline in stiffness during digestion positively correlated with the increase in equivalent alveolar diameters and negatively correlated with the nonlinearity index. These results suggest that the decline in stiffness results from rupture of collagen followed by load transfer and subsequent rupture of alveolar walls. This study may provide new understanding of the role of collagen degradation in general tissue remodeling and disease progression.

Homeostatic maintenance via degradation and repair of elastic fibers under tension.

Cellular maintenance of the extracellular matrix requires an effective regulation that balances enzymatic degradation with the repair of collagen fibrils and fibers. Here, we investigate the long-term maintenance of elastic fibers under tension combined with diffusion of general degradative and regenerative particles associated with digestion and repair processes. Computational results show that homeostatic fiber stiffness can be achieved by assuming that cells periodically probe fiber stiffness to adjust the production and release of degradative and regenerative particles. However, this mechanism is unable to maintain a homogeneous fiber. To account for axial homogeneity, we introduce a robust control mechanism that is locally governed by how the binding affinity of particles is modulated by mechanical forces applied to the ends of the fiber. This model predicts diameter variations along the fiber that are in agreement with the axial distribution of collagen fibril diameters obtained from scanning electron microscopic images of normal rat thoracic aorta. The model predictions match the experiments only when the applied force on the fiber is in the range where the variance of local stiffness along the fiber takes a minimum value. Our model thus predicts that the biophysical properties of the fibers play an important role in the long-term regulatory maintenance of these fibers.

Fluctuation-driven mechanotransduction regulates mitochondrial-network structure and function.

Cells can be exposed to irregular mechanical fluctuations, such as those arising from changes in blood pressure. Here, we report that ATP production, assessed through changes in mitochondrial membrane potential, is downregulated in vascular smooth muscle cells in culture exposed to monotonous stretch cycles when compared with cells exposed to a variable cyclic stretch that incorporates physiological levels of cycle-by-cycle variability in stretch amplitude. Variable stretch enhances ATP production by increasing the expression of ATP synthase's catalytic domain, cytochrome c oxidase and its tyrosine phosphorylation, mitofusins and PGC-1α. Such a fluctuation-driven mechanotransduction mechanism is mediated by motor proteins and by the enhancement of microtubule-, actin- and mitochondrial-network complexity. We also show that, in aorta rings isolated from rats, monotonous stretch downregulates-whereas variable stretch maintains-physiological vessel-wall contractility through mitochondrial ATP production. Our results have implications for ATP-dependent and mechanosensitive intracellular processes.

Scale dependence of structure-function relationship in the emphysematous mouse lung.

The purpose of this study was to determine how the initial distribution of elastase in mouse lungs determines the time course of tissue destruction and how structural heterogeneity at different spatial scales influences lung function. We evaluated lung function and alveolar structure in normal and emphysematous C57BL/6 mice at 2 and 21 days following orotracheal treatment with porcine pancreatic elastase (PPE). Initial distribution of elastase 1 h after treatment was assessed using red fluorescently labeled PPE (f-PPE) by laser scanning confocal microscopy. From measured input impedance of the respiratory system, the global lung compliance, and the variability of regional compliance were obtained. Lungs were fixed and equivalent airspace diameters were measured in four lobes of the right lung and three regions of the left lung. At day 2 and day 21, the mean airspace diameter of each region was significantly enlarged which was accompanied by an increased inter-regional heterogeneity. The deposition of f-PPE on day 0 was much more heterogeneous than the inter-regional diameters at both day 2 and day 21 and, at day 21, this reached statistical significance (p < 0.05). Microscale heterogeneity characterized by the overall variability of airspace diameters correlated significantly better with compliance than macroscale or inter-regional heterogeneity. Furthermore, while the spatial distribution of the inflammatory response does not seem to follow that of the elastase deposition, it correlates with the strongest regional determinant of lung function. These results may help interpret lung function decline in terms of structural deterioration in human patients with emphysema.

Changes in respiratory elastance after deep inspirations reflect surface film functionality in mice with acute lung injury.

Pulmonary surfactant reduces surface tension in the lung and prevents alveolar collapse. Following a deep inspiration (DI), respiratory elastance first drops then gradually increases due to surface film and tissue viscoelasticity. In acute lung injury (ALI), this increase is faster and governed by alveolar collapse due to increased surface tension. We hypothesized that the rate of increase in elastance reflects the deficiency of surfactant in the lung. To test this, mice were ventilated before (baseline) and after saline lavage obtained by injecting 0.8 ml and withdrawing 0.7 ml fluid (severe ALI) or injecting 0.1 ml (mild ALI). After two DIs, elastance was tracked for 10 min followed by a full lavage to assess surfactant proteins B (SP-B) and C (SP-C) content. Following 2 DIs, the increases in elastance during 10 min ventilation (ΔH) were 3.60 ± 0.61, 5.35 ± 1.04, and 8.33 ± 0.84 cmH2O/ml in baseline mice and mice with mild and severe ALI, respectively (P < 0.0001). SP-B and SP-C in the lavage fluid dropped by 32.4% and 24.9% in the mild and 50.4% and 39.6% in the severe ALI, respectively. Furthermore, ΔH showed a strong negative correlation with both SP-B (r(2) = 0.801) and SP-C (r(2) = 0.810) content. The ΔH was, however, much smaller when the lavage fluid also contained exogeneous SP-B and SP-C. Thus ΔH can be interpreted as an organ level measure of surface film functionality in lavage-induced ALI in mice. This method could prove useful in clinical situations such as diagnosing surfactant problems, monitoring recovery from lung injury or the effectiveness of surfactant therapy.