Monomolecular surface film and tubular myelin figures of the pulmonary surfactant in hamster lung

Summary Perfusion fixation via pulmonary trunk was applied to the alveolar lining layer in situ at different lung volumes using a fixative containing tannic acid-ferrocyanide osmium. The monomolecular surface film and hypophasic tubular myelin figures were enhanced. In the range of transpulmonary pressure (1–10 cmH₂O), the surface film appeared in the form of a single, electron-dense leaflet, 2.7±0.6 nm (M±SD) in thickness while trilaminar membrane structure was retained in all parts of the tubular myelin figures of the hypophase. The surface film was attached underneath at right angles with trilaminar membranes which formed the outermost parts of the tubular myelin. Such structural continuity was taken to support a view that the phospholipid unit membrane of the tubular myelin figure would be transformed at the hydrophobic phase into a pair of monomolecular leaflets, eventually forming the surface film.     

Constrained sessile drop as a new configuration to measure low surface tension in lung surfactant systems.

Existing methodology for surface tension measurements based on drop shapes suffers from the shortcoming that it is not capable to function at very low surface tension if the liquid dispersion is opaque, such as therapeutic lung surfactants at clinically relevant concentrations. The novel configuration proposed here removes the two big restrictions, i.e., the film leakage problem that is encountered with such methods as the pulsating bubble surfactometer as well as the pendant drop arrangement, and the problem of the opaqueness of the liquid, as in the original captive bubble arrangement. A sharp knife edge is the key design feature in the constrained sessile drop that avoids film leakage at low surface tension. The use of the constrained sessile drop configuration in conjunction with axisymmetric drop shape analysis to measure surface tension allows complete automation of the setup. Dynamic studies with lung surfactant can be performed readily by changing the volume of a sessile drop, and thus the surface area, by means of a motor-driven syringe. To illustrate the validity of using this configuration, experiments were performed using an exogenous lung surfactant preparation, bovine lipid extract surfactant (BLES) at 5.0 mg/ml. A comparison of results obtained for BLES at low concentration between the constrained sessile drop and captive bubble arrangement shows excellent agreement between the two approaches. When the surface area of the BLES film (0.5 mg/ml) was compressed by about the same amount in both systems, the minimum surface tensions attained were identical within the 95% confidence limits.     

Evolution of the lung surfactant proteins in birds and mammals

Phylogenetic analyses of the families of mammalian lung surfactant proteins (SP-A, SP-B, SP-C, and SP-D) supported the hypothesis that these proteins have diverged between birds and mammals as a result of lineage-specific gene duplications and deletions. Homologs of mammalian genes encoding SP-B, SP-C, and SP-D appear to have been deleted in chickens, whereas there was evidence of avian-specific duplications of the genes encoding SP-A and presaposin. Analysis of the genes closely linked to human SP-B, SP-C, and SP-D genes revealed that all three of these genes are closely linked to genes having orthologs on chicken chromosome 6 and also to genes lacking chicken orthologs. These relationships suggest that all of the lung surfactant protein genes, as well as certain related genes, may have been linked in the ancestor of humans and chickens. Further, they imply that the loss of surfactant protein genes in the avian lineages formed part of major genomic rearrangement events that involved the loss of other genes as well. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dynamic surface tension of natural surfactant extract under superimposed oscillations

Surfactant dysfunction plays a major role in respiratory distress syndrome (RDS). This research seeks to determine whether the use of natural surfactant, Curosurf? (Cheisi Farmaceutici, Parma, Italy), accompanied with pressure oscillations at the level of the alveoli can reduce the surface tension in the lung, thereby making it easier for infants with RDS to maintain the required level of functional residual capacity (FRC) without collapse. To simulate the alveolar environment, dynamic surface tension measurements were performed on a modified pulsating bubble surfactometer (PBS) type device and showed that introducing superimposed oscillations about the tidal volume excursion between 10 and 70 Hz in a surfactant bubble lowers interfacial surface tension below values observed using tidal volume excursion alone. The specific mechanisms responsible for this improvement are yet to be established; however it is believed that one mechanism may be the rapid transient changes in the interfacial area increase the number of interfacial binding sites for surfactant molecules, increasing adsorption and diffusion to the interface, thereby decreasing interfacial surface tension. Existing mathematical models in the literature reproduce trends noticed in experiments in the range of breathing frequencies only. Thus, a modification is introduced to an existing model to both incorporate superimposed pressure oscillations and demonstrate that these may improve the dynamic surface tension in the alveoli.     

Improved lung preservation relates to an increase in tubular myelin-associated surfactant protein A

Background

Declining levels of surfactant protein A (SP-A) after lung transplantation are suggested to indicate progression of ischemia/reperfusion (IR) injury. We hypothesized that the previously described preservation-dependent improvement of alveolar surfactant integrity after IR was associated with alterations in intraalveolar SP-A levels.

Methods

Using immuno electron microscopy and design-based stereology, amount and distribution of SP-A, and of intracellular surfactant phospholipids (lamellar bodies) as well as infiltration by polymorphonuclear leukocytes (PMNs) and alveolar macrophages were evaluated in rat lungs after IR and preservation with EuroCollins or Celsior.

Results

After IR, labelling of tubular myelin for intraalveolar SP-A was significantly increased. In lungs preserved with EuroCollins, the total amount of intracellular surfactant phospholipid was reduced, and infiltration by PMNs and alveolar macrophages was significantly increased. With Celsior no changes in infiltration or intracellular surfactant phospholipid amount occurred. Here, an increase in the number of lamellar bodies per cell was associated with a shift towards smaller lamellar bodies. This accounts for preservation-dependent changes in the balance between surfactant phospholipid secretion and synthesis as well as in inflammatory cell infiltration.

Conclusion

We suggest that enhanced release of surfactant phospholipids and SP-A represents an early protective response that compensates in part for the inactivation of intraalveolar surfactant in the early phase of IR injury. This beneficial effect can be supported by adequate lung preservation, as e.g. with Celsior, maintaining surfactant integrity and reducing inflammation, either directly (via antioxidants) or indirectly (via improved surfactant integrity).     

Extramembraneous particles and structural variations of tubular myelin figures in rat lung surfactant

Tubular myelin figures of pulmonary surfactant were examined by electron microscopy after fixation in glutaraldehyde and postfixation in an osmium tetroxide-ferrocyanide mixture. Bilayered membranes were seen as parallel arrays or as lattices with spacings varying from about 36 to 50 nm. This method also produced good visualization of drumstick-like particles, 5 nm in diameter and about 15 nm in length. The particles were regularly spaced at intervals of 16 nm in rows along the rectangular angles of myelin membranes. Depending on the size of the tubules the particles contacted each other in the center of the tubules at low diameters (tubular diameter less than 40 nm) and formed a continuous filamentous central core, or they were separated from one another (tubular diameter greater than 40 nm). In the latter case the central core had a hollow appearance. Based on further findings employing tannic acid, lipid extraction with 2,2-dimethoxypropane, and a ruthenium red-osmium tetroxide technique for the demonstration of polyanionic proteins it is suggested that these particles are protein in nature and that they are involved in the formation and maintenance of the structure of tubular myelin. A new concept of the ultrastructure of tubular myelin figures is proposed.     

Effect of surface tension and surfactant administration on Eustachian tube mechanics.

Development of otitis media has been related to abnormal Eustachian tube (ET) mechanics. ET is a collapsible tube that is periodically opened to regulate middle ear pressure and to clear middle ear fluid into the nasopharynx. The ability to perform these physiological functions depends on several mechanical properties, including the ET's opening pressure (P(open)), compliance (ETC), and hysteresis (eta). In this study, a previously developed modified force-response protocol was used to determine ET mechanical properties after experimental manipulation of the mucosal surface condition. Specifically, these properties were measured in the right ear of six cynomologous monkeys under baseline conditions after "washing out" the normal ET mucous layer and after instillation of a pulmonary surfactant, Infasurf. Removal of the normal mucosa did not significantly alter P(open) but did result in a decrease in ETC and eta (P < 0.05). Treatment of the mucosa with Infasurf was effective in reducing P(open) and increasing both ETC and eta to baseline values (P < 0.05). These results indicate that the mucosa-air surface tension can affect the overall ETC and eta properties of the ET. In addition, this study indicates that surfactant therapy may only be beneficial in patients with rigid or inelastic ETs (large P(open) and low ETC and eta).     

The late asthmatic response is linked with increased surface tension and reduced surfactant protein B in mice

Pulmonary surfactant dysfunction may significantly contribute to small airway obstruction during the asthmatic response, but neither its exact role nor its regulation is clear. Surfactant function and composition was studied in an Aspergillus fumigatus (Af)-induced late-phase allergic airway response in sensitized BALB/c mice. The peak of Af-induced airway hyperresponsiveness in sensitized and challenged mice 24 h after allergen provocation coincided with a significant fall in surface activity of the pulmonary surfactant. The underlying changes included time-dependent elaboration of eotaxin and IL-5 followed by eosinophil influx into the airways. The height of airway inflammation and hyperresponsiveness was preceded by release of IL-4 and marked reductions in surfactant protein (SP)-B, a hydrophobic surfactant protein responsible for maintaining low surface tension of the lining fluid of distal air spaces. Furthermore, intratracheal administration of IL-4 significantly inhibited SP-B, indicating a regulatory role of this cytokine in the surfactant biophysical changes. Thus surfactant dysfunction induced by an IL-4-driven SP-B deficiency after allergen provocation may be an important part of the late asthmatic airway response.     

Pulmonary clearance of inhaled 99mTc-DTPA: effects of surfactant depletion by lung lavage

The influence of surfactant depletion on clearance from the lungs of inhaled technetium-99m-labeled diethylenetriamine pentaacetate (99mTc-DTPA) was studied in rabbits. Surfactant was removed by repeated lung lavage with isotone saline. To minimize structural damage to the lungs, pressure generated insufflation with short expiration was utilized. Aerosolized 99mTc-DTPA was administered via a bag-in-bottle system. Radioactivity was measured with a gamma camera and time-activity curves were obtained over the base of the right lung. Six nonlavaged rabbits served as controls. In six lavaged rabbits clearance of 99mTc-DTPA was significantly faster than in controls. In three rabbits given natural surfactant into the trachea after lung lavage, 99mTc-DTPA was eliminated faster than in controls but slower than in surfactant-depleted animals. The results indicate a role of surfactant on clearance rate of 99mTc-DTPA from rabbit lungs. Measurements of 99mTc-DTPA clearance may be useful in studying the function of the surfactant system in different lung disorders.     

Proteolipid in bovine lung surfactant: its role in surfactant function

The chemical and biophysical properties of the proteins in the lipid extracts of lung surfactant have not clearly been determined. These proteins were isolated from lung surfactant lipids by Sephadex LH-20 chromatography and purified with silicic acid chromatography followed by dialysis against organic solvents. The proteolipid thus obtained had a protein to phospholipid ratio of 3 to 1 (w/w). The proteolipid apoprotein had a nominal molecular weight of ca. 5 kDa. We evaluated the functional role of this proteolipid by combining it with proteolipid-depleted surfactant lipids or synthetic dipalmitoylphosphatidylcholine (DPPC) and then measuring with a pulsating bubble surfactometer. The proteolipid and DPPC recombinant reproduced the surface activity of natural lung surfactant. We conclude that this 5 kDa proteolipid apoprotein is a functionally important constituent of lung surfactant.