Lung expansion and survival in rabbit neonates treated with surfactant extract

Preterm rabbit fetuses, delivered on the 27th day of gestation, were studied following upper airway instillation with either natural surfactant (NSA) obtained from the lavage of adult rabbit lungs or with a protein-free suspension of lipids extracted from lung wash (ESA). First, lung compliance was studied postmortem. The administration of 25 microliters of either preparation resulted in greater hysteresis (P less than 0.05) than was observed in control fetuses receiving no surfactant material. Increasing the phospholipid concentration stepwise from 10 to 50 mg/ml improved airway expansion and stability. No further improvement was encountered with concentrations greater than 50 mg/ml. There was no significant difference in compliance response between NSA and ESA. Morphometry of the lungs also indicated that the two preparations had an equal effect on compliance. Second, it was determined how neonatal survival was affected by a pharyngeal deposition, prior to the first breath, of 50 microliters NSA or ESA. Both treatment groups demonstrated improved survival (P less than 0.001) when compared with controls receiving no pharyngeal deposition. These findings offer further support to the concept that protein is not required for the efficacy of a surfactant supplementation. The equivalence of the two preparations suggests that a sterile suspension of a protein-free surfactant extract could be used to prevent or treat respiratory distress in preterm neonates.     

A synthetic surfactant based on a poly-Leu SP-C analog and phospholipids: effects on tidal volumes and lung gas volumes in ventilated immature newborn rabbits.

Available surfactants for treatment of respiratory distress syndrome in newborn infants are derived from animal lungs, which limits supply and poses a danger of propagating infectious material. Poly-Val-->poly-Leu analogs of surfactant protein (SP)-C can be synthesized in large quantities and exhibit surface activity similar to SP-C. Here, activity of synthetic surfactants containing a poly-Leu SP-C analog (SP-C33) was evaluated in ventilated premature newborn rabbits. Treatment with 2.5 ml/kg body wt of 2% (wt/wt) SP-C33 in 1,2-dipalmitoyl-sn-3-glycero phosphoryl choline (DPPC)-1-palmitoyl-2-oleoyl-sn-3-glycero phosphoryl choline (POPC)-1-palmitoyl-2-oleoyl-sn-3-glycero phosphoryl glycerol (POPG), 68:0:31, 68:11:20, or 68:16:15 (wt/wt/wt) suspended at 80 mg/ml gave tidal volumes (Vt) of 20-25 ml/kg body wt, with an insufflation pressure of 25 cmH2O and no positive end-expiratory pressure (PEEP), comparable to the Vt for animals treated with the porcine surfactant Curosurf. Nontreated littermates had a Vt of approximately 2 ml/kg body wt. The Vt for SP-C33 in DPPC-egg phosphatidylglycerol-palmitic acid [68:22:9 (wt/wt/wt)], DPPC-POPG-palmitic acid [68:22:9 (wt/wt/wt)], and DPPC-POPC-POPG [6:2:2 (wt/wt/wt)] was 15-20 ml/kg body wt. Histological examination of lungs from animals treated with SP-C33-based surfactants showed incomplete, usually patchy air expansion of alveolar spaces associated with only mild airway epithelial damage. Lung gas volume after 30 min of mechanical ventilation were more than threefold larger in animals treated with Curosurf than in those receiving SP-C33 in DPPC-POPC-POPG, 68:11:20. This difference could be largely counterbalanced by ventilation with PEEP (3-4 cmH2O). An artificial surfactant based on SP-C33 improves Vt in immature newborn animals ventilated with standardized peak pressure but requires PEEP to build up adequate lung gas volumes.     

Lung pathogenesis. VIII. Relationships between alveolar surfactant disorders and development of lung diseases

The new data concerning the structure and dynamics of the alveolar surfactant, its phospholipid and apoprotein components and their synthesis, storage and secretion by the large granular alveolocytes, the formation and disconnection of lipoprotein complexes, their disorders and pathological relationships were analysed in order to discern the possibility of a more or less important pathogenetic role in the onset and development of lung diseases. Sometimes, surfactant disorders appeared as epiphenomena, like in pulmonary edemas; at other times, they behaved as a turn plate enhancing and centering the development of alveolar lipoproteinosis, microlithiasis alveolaris, hyaline membrane disease of newborns and the adult respiratory distress syndrome. Focusing the pathogenesis on the surfactant disorders and on their causes, a unification of mechanisms became possible with the increase in complexity of processes by the intervention of other complicating factors, mainly the self-perpetuating ones.     

Effects of various forms of surfactant protein C on tidal volume in ventilated immature newborn rabbits.

Surfactant protein (SP)-C is characterized by alpha-helix structure and palmitoyl groups attached to two cysteine residues. We examined the function of palmitoylation and dimerization in promotion of tidal volume in immature newborn rabbits. Reconstituted surfactants were made from a mixture of synthetic phospholipids and porcine SP-B (basic mixture) by adding various forms of SP-Cs: normal SP-C isolated from porcine lungs and monomeric or dimeric forms of SP-C. These latter two were isolated from patients with pulmonary alveolar proteinosis and were less palmitoylated. Animals were ventilated at an inspiratory pressure of 25 cmH2O. Median tidal volumes were <2 ml/kg in nontreated controls, 7.7 ml/kg in animals receiving the basic mixture without SP-C, and >18 ml/kg in animals treated with reconstituted surfactants containing 3% normal or 2% dimeric SP-C (P < 0.05 vs. basic mixture). The physiological effect of basic mixture was not improved by monomeric SP-C. We conclude that palmitoyl groups are important for the physiological effects of SP-C and that the dimeric form also improves physiological effects.     

Lung surfactant protein A (SP-A) enhances serum-independent phagocytosis of bacteria by alveolar macrophages.

Surfactant protein A (SP-A) is the main protein component of lung surfactant. We studied the involvement of SP-A in body defense, i.e., effect of SP-A on the phagocytosis of bacteria by alveolar macrophages. We show here that SP-A enhances the phagocytosis of some non-opsonized bacteria: Escherichia coli growing logarithmically (E. coli/log), Pseudomonas aeruginosa/log as well as from stationary phase (P. aeruginosa/stat) and Staphylococcus aureus/log. Furthermore, not only serum-independent phagocytosis was effected by SP-A but also phagocytosis of serum-opsonized S. aureus/stat. No effect of SP-A on phagocytosis was observed with E. coli/stat neither on serum-independent nor on serum-dependent phagocytosis and on phagocytosis of non-opsonized S. aureus/stat. Thus, effect of SP-A on phagocytosis is dependent on bacterial species and on the growth phase of the microorganisms, and this effect is concentration dependent. We studied two different human recombinant SP-As and SP-A isolated from lung lavage material from proteinosis patients. These SP-A molecules contain different isomeric chains, and they differ in complexity of their structure. Qualitatively, we found the same effect with all three substances. Quantitatively, the proteinosis SP-A that forms the most complex structure was the most effective. Taken together, we demonstrated a stimulating effect of SP-A on serum-independent as well as on serum-dependent phagocytosis of bacteria by alveolar macrophages, both depending on species and growth phase of the bacteria.     

Lipids in the lungs and alveolar surfactant in anaphylactic shock

The process of anaphylactoid response of rats to introduction of egg protein is associated with a decrease of the pulmonary surfactant surface activity. The factors of metabolic surfactant inactivation are as follows: protein accumulation, the disturbance of lipids transport between pulmonary cells and alveolar surface, change in fatty-acidic composition of surfactant phospholipids. The isolation of arachidonic acid from surfactant phospholipids in anaphylactoid shock is an evidence for the participation of the pulmonary surface-active phase in the process of biosynthesis of the lipid mediators in respiratory organs.     

Reutilization of surfactant phosphatidylcholine in adult rabbits

32P-saturated phosphatidylcholine was added to [3H]choline-labeled natural surfactant and the mixture was injected intratracheally into 87 adult rabbits. The rabbits were also given [14C]palmitate intravenously at the same time. Rabbits were killed in groups from 10 min to 72 h after injection. In each rabbit we measured the total recovered [3H]phosphatidylcholine (PC) in the alveolar wash, the ratio of [3H]PC to [32P]PC in the alveolar wash, and the specific activity of [14C]PC in the alveolar wash and lamellar bodies. Values were averaged for all rabbits killed at the same times and smooth curves were fit to the data by computer. From the intravenous [14C]palmitate data we calculated a turnover time for alveolar PC of 6.0 h. From the intratracheal labeling data, we calculated a turnover time for alveolar PC of 5.7 h and determined that alveolar PC was reutilized at an efficiency of only 23%. We also concluded that this reutilization occurred as intact molecules.     

Heterogeneity of immunohistochemical staining with pulmonary surfactant protein A among fractionated alveolar macrophages which involves metabolism of pulmonary surfactant.

Alveolar macrophages (AM) which are separated into four fractionated subpopulations (I, II, III and IV), represented differential immunohistochemical staining with antibody against pulmonary surfactant protein A (SP-A). In light microscopy, the least dense AM (fraction I) were intensely stained with antibody to SP-A in numerous granules of the cytoplasm, whereas the most dense cells (fraction IV) showed immuno-reactivity with the antibody in several granules distributed in the spreading and elongating cytosol. By Western blot analysis, antibody to SP-A recognized a triplet of nature molecules of SP-A in AM lysate. However, the antigen of the AM lysate almost disappeared when the cells were cultured for more than two days, which indicate that AM do not synthesize SP-A and have digested intracellular SP-A during the cultivation. Immunoelectron microscopically, AM of fraction IV sometimes had very large vacuoles including lamellar body-like structures, probably pulmonary surfactant immediately after taken up from the alveolar lumen by them, which were heavily deposited with gold particles indicating antigenic site of SP-A. Whereas cells of fraction I contained numerous cytoplasmic vacuoles that were frequently labelled with the immuno-gold particles and were not associated with lamellar body-like structures, which may indicate that the materials in the vacuoles are digesting. The results of this experiments suggest that pulmonary surfactant, layered on the alveolar epithelium, is in part taken up by higher dense AM and is digested during a process of their maturation in the direction of lower dense cells, which undergo an important role of metabolism of pulmonary surfactant by AM subpopulations.     

Lung volumes, mechanics, and single-breath diffusing capacity in anesthetized cats.

We measured lung weight, lung volumes, pulmonary mechanics, and carbon monoxide transfer (DLCO, single-breath method) in healthy cats (3.3 +/- 0.4 kg) that were anesthetized, paralyzed, and mechanically ventilated through a tracheal cannula. Compared with Stahl's predicted values which were based on regression analyses of data collected from several species, our cats had larger and more compliant lungs in relation to body weight, higher DLCO per unit body weight, and similar DLCO/TLC (size independent constant). Compared with Robinson et al.'s values derived entirely from studies on dogs, our cats had significantly smaller lung volumes and DLCO per unit body weight, DLCO/TLC and similar ratios of CL/FRC. Several factors appear to contribute to the functional variations among mammalian species: differences in the relation of lung to body weight, differences in the relation of chest wall compliance to lung compliance, and differences in the fundamental structure and design of the respiratory systems. Differences in methodology are acknowledged to be an additional factor.     

Increased surfactant protein A content in human alveolar macrophages in hypersensitivity pneumonitis.

Surfactant protein A (SP-A) appears to have an important function in the assembly and maintenance of the alveolar surfactant monolayer. SP-A has also been implicated in modulating the activity of immunoactive cells, such as increasing the bactericidal capacity of alveolar macrophages. In this immunocytochemical study the SP-A content of alveolar macrophages from seven patients with hypersensitivity pneumonitis was compared with the results obtained from six healthy controls. A polyclonal rabbit antibody against human SP-A was used for detection of SP-A in the cytoplasm of alveolar macrophages, applying the immunoperoxidase adhesive slide assay. In hypersensitivity pneumonitis a significant increase in the percentage of SP-A+ alveolar macrophages was observed as compared with the percentage in healthy controls. The intensity of the staining reaction was also increased in the alveolar macrophages of hypersensitivity pneumonitis. We conclude that the observed abnormalities in SP-A content in alveolar macrophages may play a role in the pathogenesis of hypersensitivity pneumonitis.     

Lung volumes and respiratory mechanics in elastase-induced emphysema in mice

Absolute lung volumes such as functional residual capacity, residual volume (RV), and total lung capacity (TLC) are used to characterize emphysema in patients, whereas in animal models of emphysema, the mechanical parameters are invariably obtained as a function of transrespiratory pressure (Prs). The aim of the present study was to establish a link between the mechanical parameters including tissue elastance (H) and airway resistance (Raw), and thoracic gas volume (TGV) in addition to Prs in a mouse model of emphysema. Using low-frequency forced oscillations during slow deep inflation, we tracked H and Raw as functions of TGV and Prs in normal mice and mice treated with porcine pancreatic elastase. The presence of emphysema was confirmed by morphometric analysis of histological slices. The treatment resulted in an increase in TGV by 51 and 44% and a decrease in H by 57 and 27%, respectively, at 0 and 20 cmH(2)O of Prs. The Raw did not differ between the groups at any value of Prs, but it was significantly higher in the treated mice at comparable TGV values. In further groups of mice, tracheal sounds were recorded during inflations from RV to TLC. All lung volumes but RV were significantly elevated in the treated mice, whereas the numbers and size distributions of inspiratory crackles were not different, suggesting that the airways were not affected by the elastase treatment. These findings emphasize the importance of absolute lung volumes and indicate that tissue destruction was not associated with airway dysfunction in this mouse model of emphysema.     

Donor lung pretreatment with surfactant in experimental transplantation preserves graft hemodynamics and alveolar morphology

In experimental lung transplantation, the reduction of endogenous surfactant properties occurs after graft preservation and transplant reperfusion. The aim of this study was to evaluate the efficacy of donor lung pretreatment with exogenous surfactant on graft damage after ischemia and reperfusion. Fourteen (control group A, n = 8; study group B, n= 6) young female white pigs (mean weight 27 +/- 3.5 kg) were used in a newly developed autotransplantation model within situcold ischemia. In study group B, before thoracotomy, 1.5 ml/kg surfactant apoprotein-A-free surfactant was administrated into the left main bronchus via flexible bronchoscopy. Belzer UW solution was used for lung preservation. Cold ischemia was achieved for 3 hr with interlobar lung parenchyma temperature at 8 +/- 1.3 degrees C, and central temperature maintained at 37.20 +/- 0.5 degrees C. Animals were sacrificed after 3 hr of graft reperfusion. At the end of reperfusion, pulmonary vascular resistance index (was 447.80 dyn/sec.cm(5).m(2)(+/-66.8) in group A vs 249.51 in group B (P< 0.001) and serum nitric oxide was adequately preserved. The mean alveolar surface area estimated by computerized morphometry was 5280.84 (4991.1) microm(2)(group A) vs 3997.89 (3284.70) microm(2)(group B;P< 0.005). Histology revealed milder macrophage and lymphocyte infiltration in group B at the end of reperfusion. Pretreatment of donor lung with an surfactant apoprotein-A -free surfactant agent appears to be beneficial in terms of maintaining serum NO and reducing hemodynamic disturbances. Furthermore, alveolar histology and stereomorphology are better preserved.     

Infection with and antibody response to Pasteurella multocida and Bordetella bronchiseptica in immature rabbits

At 2, 4, 6, 8 and 10 weeks of age rabbits were cultured for Pasteurella multocida and Bordetella bronchiseptica from the paranasal sinuses, trachea, middle ears, lungs and liver. Sera were tested for antibodies (IgG) against P. multocida and B. bronchiseptica. Pasteurella multocida was isolated from all ages of rabbits, and B. bronchiseptica was isolated from rabbits 4 to 10 weeks old. The sinuses were colonized most often, followed by the trachea, middle ears and lungs. No bacteria were isolated from the liver. The number of rabbits with antibodies against both bacteria decreased between 2 and 6 weeks of age, indicating a fall in maternal antibodies, and increased between 6 and 8 weeks of age, suggesting an active humoral response.