Hans Bachofen

FORMATION AND STRUCTURE OF SURFACE FILMS: CAPTIVE BUBBLE SURFACTOMETRY

The adsorption model for soluble surfactants has been modified for suspensions of pulmonary surfactant. The dynamic adsorption behavior may be governed by a two-step process: (1) the transfer of molecules between the surface layer and the subsurface layer, which has a thickness of a few molecular diameters only; (2) the exchange of molecules between the subsurface and the bulk solution. The first step is an adsorption process and the second step is a mass transfer process. Between the subsurface and the bulk solution is an undisturbed boundary layer where mass transport occurs by diffusion only. The thickness of this boundary layer may be reduced by stirring. Rapid film formation by adsorption bursts from lipid extract surfactants, as observed in the captive bubble system, suggests that the adsorption process as defined above is accompanied by a relatively large negative change in the free energy. This reduction in the free energy is provided by a configurational change in the association of the specific surfactant proteins and the surfactant lipids during adsorption. The negative change in the free energy during film formation more than compensates for the energy barrier related to the film surface pressure. In the traditional view, the extracellular alveolar lining layer is composed of two parts, an aqueous subphase and a surfactant film, believed to be a monolayer, at the air-water interface. The existence and continuity of the aqueous subphase has recently been demonstrated by Bastacky and coworkers, and a continuous polymorphous film has recently been shown by Bachofen and his associates, using perfusion fixation of rabbit lungs with slight edema. In the present chapter, we have described a fixation technique using a non-aqueous fixation medium of perfluorocarbon and osmium tetroxide to fix the peripheral airspaces of guinea pig lungs. A continuous osmiophilic film which covers the entire alveolar surface, including the pores of Kohn, is demonstrated. By transmission electron microscopy, the surface film frequently appears multilaminated, not only in the alveolar corners or crevices, but also at the thin air-blood barrier above the capillaries. Disk-like structures or multilamellar vesicles appear partially integrated into the planar multilayered film. In corners and crevices, tubular myelin appears closely associated with the surface film. Tubular myelin, however, is not necessary for the generation of a multilaminated film. This is demonstrated in vitro by the fixation for electron microscopy of a film formed from lipid extract surfactant on a captive bubble. Films formed from relatively high surfactant concentration (1 mg/ml of phospholipid) are of variable thickness and frequent multilayers are seen. In contrast, at 0.3 mg/ml, only an amorphous film can be visualized. Although near zero minimum surface tensions can be obtained for both surfactant concentrations, film compressibility and mechanical stability are substantially better at the higher concentrations. This appears to be related to the multilaminated structure of the film formed at the higher concentration.

Alveolar surface tensions in excised rabbit lungs: Effect of temperature

In excised, perfused rabbit lungs the alveolar surface tension was measured in individual alveoli over the entire P-V loop at different temperatures (22 and 37 degrees C), using an improved microdroplet method. Additional in vitro experiments are reassuring that the microdroplets do not affect the properties of the alveolar surface film. The in situ measurements show that the alveolar surface tension and the surface tension to volume relation are essentially the same at 22 and 37 degrees C. A maximal surface tension of about 30 mN X m-1 was measured at TLC, and there is a substantial surface tension to volume hysteresis, which amounts to almost 10 mN X m-1 in the middle volume range of a complete pressure volume cycle of the lung. However, with respect to the absolute values of alveolar surface tension, and the shape and width of the hysteresis, these directly obtained results are different from previous findings.

Adaptation of the growing lung to increased V̇O2: III. The effect of exposure to cold environment in rats

This study was undertaken to further test the hypothesis that increased Vo2 operates as a stimulus for enhanced lung growth leading to a pulmonary diffusing capacity adapted to the bodys O2 requirements. Vo2 was augmented by raising 4-week-old rats for 3 weeks at 11 degrees C ambient temperature, with controls kept at 24 degrees C; this led to an increase in Vo2 averaged over 24 h by 64%. In contrast to previous experiments with waltzing mice this regime did not affect body growth, as the final body weights were identical in both groups. In the cold-exposed rats the lung volume was larger by 24%, due to an increase by 26% in air volume (at about TLC), 13% in capillary blood volume and 19% in tissue volume. The alveolar and capillary surface areas were increased by 18%, and Dm and Dl by 17% and 21% respectively. It is concluded that the hypothesis of adaptation of pulmonary gas exchange capacity to increased Vo2 cannot be rejected. Whilst in previous experiments some doubts had to be retained as to the specificity of the stimulus, because of its rather marked effect on body weight, this reservation does not hold in this case. The structural modifications which lead to increased Dl in the various experimental models are discussed.

Lung Lesions in Experimental Hydrostatic Pulmonary Edema: An Electron Microscopic and Morphometric Study

Distinct barrier lesions and an apical-basal distribution of alveolar edema fluid in either moderate or high elevated pressure edema lungs have been found in previous studies. In the present study, quantitative measurements were obtained by using electron microscopy and morphometry of extravascular lung water and barrier lesions, on the relations between interstitial and alveolar edema fluid as well as between extravascular lung water and barrier lesions. The study further addressed the question of whether 6% bovine serum albumin (BSA) perfusion could induce lung ultrastructure alterations. It was found that interstitial fluid distribution is similar to that of alveolar edema fluid. Epithelial blebs are also distributed with an apical-basal gradient, and are always submerged in alveolar edema fluid. Perfusion with 6% bovine serum albumin does not induce any lung ultrastructure alterations. The results indicate that endothelium and epithelium play a different role in controlling fluid movement between capillary and extravascular spaces and thus in preventing the formation of interstitial and alveolar edema. Because the interaction of cells and tissue must be taken into account, simple physiological models of pulmonary fluid exchange may not be adequate to explain pulmonary edema formation.

Lung fixation by airway instillation: effects on capillary hematocrit.

Instillation of glutaraldehyde into the airways of collapsed lungs is a widely used fixation procedure for analysis of pulmonary structure-function relations. In order to better define the effect of this fixation method on the pulmonary microvasculature, a morphometric analysis was made in rat lungs fixed under different conditions. In one group of animals the lungs were allowed to collapse; then the circulation was suddenly interrupted with a sling placed in the coronary sulcus before instillation of the fixative. In a second group the same procedure was carried out with the exception that the lungs were kept inflated at the time of circulatory arrest. In third group the standard technique was used, i.e., the collapsed lungs were instilled before circulatory arrest. The results show that important parameters of alveolar and septal dimensions, and in particular the capillary surfaces and volumes, are alike regardless of the procedure of fixation. However, in lungs fixed by the standard technique a higher capillary hematocrit (approximately 33%) and a decreased harmonic mean thickness of the plasma barrier were found. This artificial hemoconcentration in the microvasculature resulted in a substantial morphometric overestimate of the diffusing capacity of the lung.

Morphometric estimates of diffusing capacity in lungs fixed under zone II and zone III conditions

Comparative morphometric estimates of the diffusing capacity (DL) were made in rabbit lungs fixed by vascular perfusion under lower zone II and zone III conditions and in lungs fixed by instillation of fixatives into the airways. Owing to a reduction of both capillary volume and membrane diffusing capacity DL of zone II lungs (0.074 +/- 0.007 (SD) ml . sec-1 . mbar-1). was found to be lower by some 25% than DL of instillation-fixed lungs (0.102 +/- 0.012 (SD) ml . sec-1 . mbar-1). The average value of DL of air-filled zone III lungs, on the other hand, almost matched the DL of instillation-fixed lungs. However, DL is not equal in all regions but increases along the vertical axis of zone III lungs. Hence, the previous conclusion that morphometric estimates of DL in instillation-fixed lungs reflects a structural limit for O2 diffusion, which cannot be reached under physiologic conditions, must be revised.

Effects of glutaraldehyde or osmium tetroxide fixation on the osmotic properties of lung cells

The osmotic properties of lung cells have been tested before and after perfusion fixation of isolated, perfused lungs with either glutaraldehyde or osmium tetroxide. The testing procedure was to add hypertonic sucrose to the perfusate for several minutes and monitor the lung weight response (an ‘osmotic transient’). Each lung was perfused with one or the other fixative solutions for 10 min, then the perfusate was changed back to Ringer‐lactate before the post‐fixation test was conducted. The results indicate that osmium tetroxide makes the cell membranes as permeable to sucrose as to water, and that sucrose thus causes no osmotic volume change. Glutaraldehyde, on the other hand, apparently preserves the impermeability of the cell membranes to sucrose, but the osmotic volume response is attenuated, indicating that significant changes in the cells have occurred.

Effect of acidosis on bilirubin-lipid extract surfactant interaction

The risk of bilirubin toxicity in newborn infants with respiratory distress syndrome and hyperbilirubinemia may depend on many factors including pH of the system. Biophysical activity and inhibition characteristics were studied in vitro for lipid extract surfactant (Curosurf, 0.25 mg/ml phospholipids), bilirubin (1.0 mg/ml dissolved in NaOH) and mixed solutions at different pH ranging from 5.0 to 7.4. It was found that unconjugated bilirubin modifies surface tension behavior of lipid extract surfactant films. Maximum and minimum surface tension levels were significantly higher in mixed solutions compared to experiments using pure Curosurf independently from pH. Film area compression for pure Curosurf was not influenced by pH and varied between 22 +/- 4% at pH 5.0 and 23 +/- 9% at pH 7.4. Adding bilirubin to lipid extract surfactant, area compression to achieve minimum surface tension increased significantly to 83 +/- 4% at pH 5.0 and 85 +/- 4% at pH 7.4 (p < 0.01). Bilirubin alone showed negligible surface activity independently from pH (83 +/- 7% at pH 5.0 and 78 +/- 9% at pH 7.4) (p > 0.5). We conclude that bilirubin has a detrimental effect on the surface tension properties of lipid extract surfactant in vitro and that this interaction is independent from pH. These data suggest no influence of acidosis on alveolar surfactant system in babies with respiratory distress syndrome and hyperbilirubinemia.