Alan J. Mautone

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.

Surfactant liquid and black foam film formation and stability in vitro and correlative conditions in vivo

Abstract We studied foam film formation by three preparations that are used as surfactant replacement therapy by injection into the lungs of neonatal infants with surfactant insufficiency (“respiratory distress syndrome”). The preparations contain either all putative hydrophobic components of the normal surfactant system (prepared from bovine lung lavage, Infasurf (IN); prepared from minced bovine lung tissue, Survanta (SU)) or a single component of the system, dipalmitoylphosphatidylcholine (Exosurf Neonatal (EX)). Foam film formation is relevant directly to normal surfactant structure and function in vivo in the neonatal period (Pediatr. Res. 12 (1978) 1070–1076). We assessed morphology and stability of liquid to black film formation and its dependence on substrate concentration C, adsorption time, temperature T, “capillary pressure” Pc, film drainage time τ0–1 and black film formation times τ1–2. We found that (1) in contrast with the other preparations, IN regularly formed stable black films at the lowest C (“threshold C” Ct) under all conditions of T (22 and 37°C) and Pc (0.3, 0.4, 1.2 and 2.4 × 103 dyn cm−2), (2) EX also formed black films but differed from IN in that Ct was higher, film integrity required a longer adsorption time and black film formation was blocked (instability) at Pc values of 1.2 and 2.4 × 103 dyn cm−2 and (3) SU did not form black films but instead formed slowly (long τ0–1 and τ1–2), at relatively high Ct, viscosity-dependent inhomogeneous atypical (“rheological”) films containing aggregates of material in the preparation. Extrapolation to in-vivo conditions of C, T and Pc indicates that both IN and SU (but not EX) may form stable foam films in situ, that film formation by IN is the more efficient process and that stable surfactant foam films, formed naturally in vivo, are consistent with both the in-vitro characteristics described here and correlative lung function in vivo.

Intraalveolar bubbles and bubble films: II. Formation in vivo through adulthood

Intraalveolar bubbles and bubble films have been shown to be part of the normal alveolar architecture in vivo from birth through the first 2 days of extrauterine life of rabbit pups (Scarpelli et al., 1996a. Anat. Rec. 244:344–357). The intraluminal boundary between airway free gas and alveolar bubbles at the level of respiratory bronchioles is established within 1 hour after birth. We now examine the lung through the rest of development, namely, 2 weeks, 1, 2, and 3 months, and adulthood.

Intranasal Metered Dose Aerosolized Surfactant Reduces Passive Opening Pressure of the Eustachian Tube: Comparison Study in Two Animal Models

Hypothesis Intranasal metered dose aerosolized inhaler surfactant will reduce opening pressure of the eustachian tube in gerbils and mice. Background Eustachian tube opening pressure necessary to allow ventilation of the middle ear must exceed the contractile force exerted by the tension on the curved surface of the eustachian tube. When the active function of opening the eustachian tube is inefficient, functional collapse of the tube persists, resulting in negative middle ear pressure. Such dysfunction is the primary cause of otitis media with effusion in children. Methods Surfactant in a metered dose aerosolized intranasal delivery system was administered to 61 healthy Mongolian gerbils and 34 albino mice. In the first arm of the experiment, measurements of eustachian tube passive opening pressure were taken at baseline, after the delivery of propellant, and then at 5 and 10 minutes after surfactant administration. In the second arm of the experiment, the animals were divided into control, propellant, and experiment groups. At zero minutes, propellant was administered to the propellant group and intranasal surfactant was administered to the experiment group. Measurements of eustachian tube passive opening pressure were taken at 0, 5, and 10 minutes. Results Surfactant administration in the gerbil reduced eustachian tube passive opening pressure from a basal measurement of 40.5 mm Hg to 32.5 mm Hg after 5 minutes and to 30.1 mm Hg after 10 minutes. In mice, eustachian tube passive opening pressure reduced from a basal level of 41.8 mm Hg to 31.3 mm Hg after 5 minutes and to 31.2 mm Hg after 10 minutes in the surfactant group. Propellant placebo resulted in no change from basal level in both animal models and in both arms of the experiment. Conclusion Intranasal metered dose inhaler aerosolized surfactant is effective in reducing eustachian tube passive opening pressure.

Dosage Regimens of Intranasal Aerosolized Surfactant on Otitis Media with Effusion in an Animal Model

OBJECTIVE: To determine optimal dosage regimens of intranasal metered dose aerosolized surfactant with and without other medications in the treatment of otitis media with effusion (OME). STUDY DESIGN: Resolution of experimental OME in gerbils was determined based on otomicroscopy and tympanometry. Experimental intranasal drugs were: surfactant, surfactant with betamethasone, surfactant with phenylephrine, and a normal saline solution placebo. Medications were administered once or twice daily via a metered dose inhaler. RESULTS: For twice-daily dosing, mean days to OME resolution were 8.5 for the aerosolized surfactant, 6.3 for the surfactant with betamethasone, 18.7 for the surfactant with phenylephrine, and 16 each for control and placebo. Resolution with the once-daily dosage was longer for all conditions. Results were comparable using tympanometry. CONCLUSION: OME resolved faster than the natural course when treated with intranasal surfactant with and without steroids. Twice-daily dosing was statistically superior. SIGNIFICANCE: This study reiterates the effectiveness of OME treatment with an aerosolized synthetic surfactant with and without steroids and establishes a superior twice-daily dosage schedule.

Intraalveolar bubbles and bubble films: I. Formation and development during the first 48 hours of extrauterine life in rabbits

Aeration of mature lungs at birth depends on formation of intraalveolar bubbles and bubble films (Scarpelli 1978. Pediatr. Res., 12:1070–1076). Bubbles establish immediately structural stability and pulmonary gas exchange. Given that air spaces are cleared in minutes of fetal liquid (the initial substrate for bubble formation), is formation possible beyond this period? If so, is this related to early development of pulmonary function and structure?

A new surface balance for dynamic surface tension studies

Abstract A new design for a dynamic surface balance is described which utilizes a vertical mesh cylinder to alter surface area. The device is described and initial studies have been carried out. Results from DPPC monolayer experiments indicate that force—area isotherms using this device closely agree with results from standard surface balances. The described surface balance has a number of advantages over the Langmuir trough such as preventing leaks in surface films, a wide frequency range and greater accuracy using a Wilhelmy plate.

Intraalveolar bubbles and bubble films: III. Vulnerability and preservation in the laboratory

Having confirmed (Scarpelli et al. 1996. Anat. Rec. 244:344‐357 and 246:245‐270) the discovery of intraalveolar bubbles and films as the normal anatomical infrastructure of aerated alveoli at all ages, we now address three questions. Why have these structures been so elusive? Visible in fresh lungs from the in vivo state, can they be preserved by known laboratory methods? Can they be preserved intact for study in tissue sections?

Intranasal phenylephrine-surfactant treatment is not beneficial in otitis media with effusion

BACKGROUND Otitis media is the most common reason for non-well-child visits to the primary care physician. Various treatments are in use to try to ameliorate the pain arising from Eustachian tube (ET) dysfunction. One such treatment is topical phenylephrine spray, despite clinical evidence disputing its use. Previous research by this laboratory has demonstrated the beneficial effect of topical surfactant treatment in reducing ET opening pressure, allowing the tube to open. This study is designed to test the effectiveness of topical phenylephrine, delivered with surfactant, in reducing days of effusion in OME, in an animal model. METHODS OME was generated by injecting Klebsiella pneumoniae lipopolysaccharide into the right bullae of 28 gerbils. After frank OME resulted, the animals were divided into four groups. Group 1 received no treatment or propellant spray alone (placebo). Group 2 received intranasal surfactant spray once daily. Group 3 received intranasal surfactant-phenylephrine spray once daily. Group 4 received intranasal surfactant-phenylephrine spray twice daily. All animals were evaluated on a daily basis by both otomicroscopy and tympanometry, and treatment was ceased when the ear returned to normal appearance. Evaluations were continued for a total of 30 days. Chi-squared analysis with significance set at .05 was performed. RESULTS In the untreated and placebo groups, middle ear effusion resolved at 16.25 days by otomicroscopy and 28.26 days by tympanometry. In Group 2 (surfactant alone), effusion resolved at 10.57 days and 15.71 days, respectively. In Group 3 (surfactant-phenylephrine once daily), effusion resolved at 15.67 days and 28.33 days. In Group 4 (surfactant-phenylephrine twice daily), effusion resolved at 18.67 days and 28.33 days. These results were statistically significant. CONCLUSIONS Intranasal phenylephrine-surfactant treatment is shown to be at least ineffective, and possibly detrimental, in the resolution of OME, in this animal model. The hypothesis is that surfactant potentiates the drying effect of phenylephrine at the ET by allowing it to get to the ET more easily; in addition, the drying effect of phenylephrine prevents full surfactant action. We believe that these results can be extrapolated to humans and that phenylephrine should be avoided in these cases.

Effect of Epinephrine Delivered Endotracheally in a Surfactant Based Aerosol via Metered Dose Inhaler (MDI) Compared to Instillation in Normal and Shock Rabbit Models • 373

We compared hemodynamic effects of two methods of administering epinephrine endotracheally in two different preparations: 1.) A 100μg/kg solution was instilled directly into the endotracheal tube; and 2.) 75μg/spray, 1 spray/kg in a surfactant-based aerosol preparation. New Zealand White rabbits(n=18) weighing 2.41 +/- 0.27 kg were anesthetized with Ketamine (55mg/kg), Xylazine (5mg/kg) and Acepromazine (0.01 mg/kg), IM, tracheostomized, and internal carotids cannulated. Blood pressure and heart rate were continuously monitored. Normal model: both epinephrine preparations were given endotracheally without any other medications either by direct instillation(n=5) with a lcc saline flush, or by aerosol (n=5). Heart rate, systolic blood pressure, diastolic blood pressure and pulse pressure were recorded pre-treatment, with treatment, then 30 sec., 1,5,10 and 20 min. thereafter.Shock model: rabbits were paralyzed with Vecuronium (0.1mg/kg) and observed until systolic blood pressure decreased to 40-45 mmHg. They were then ambu-bagged for 30 sec. and epinephrine given by direct instillation (n=4) or by aerosol (n=4). Hemodynamic parameters were recorded as above. Analysis of variance was performed on the data. Normal model: there was no significant difference between the two groups in any parameter at any time. There were significant changes over time within each groups. Shock model: there was no significant difference between the two groups except in pulse pressure at 10 minutes after treatment. (However, in both models there was a slight, but insignificant, drop in blood pressure at 30 seconds using the instilled preparation. This may be explained by a vagal response induced by the saline flush.)