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 volumes, chest wall configuration, and pattern of breathing in microgravity

We studied the changes in functional residual capacity (FRC), thoracoabdominal volume (Vw), and chest wall configuration in five normal subjects seated in an aircraft flying parabolic trajectories resulting in 20-s periods of microgravity. We measured vital capacity (VC), inspiratory capacity, and tidal volume by integrating airflow at the mouth and changes in rib cage and abdominal volume (delta Vrc and delta Vab, respectively, where delta Vrc + delta Vab = delta Vw) using induction plethysmography. During microgravity (0 Gz) FRC decreased by 413 +/- 70 (SE) ml and VC by 0.37 liter. The decrease in Vw did not differ from that in FRC and was entirely the result of reduction of Vab, the Vrc showing no significant change. During tidal breathing the abdominal contribution (delta Vab/delta Vw) increased from 0.39 +/- 0.08 at 1 Gz to 0.57 +/- 0.08 at 0 Gz. During brief periods of hypergravity (approximately 1.8 Gz) all changes were opposite in sign and relatively smaller. Limited data during "roller coaster" flight patterns suggested that, in contrast to configurational changes, the temporal pattern of breathing was uninfluenced by changes in Gz. We conclude that at the onset of weightlessness there are substantial changes in lung volume and thoracoabdominal configuration. Abdominal contribution to tidal excursions increases but the temporal pattern of breathing is unchanged.     

Convertase activity in alveolar surfactant and lamellar bodies in fetal, newborn, and adult rabbits

Conversion ofheavy-aggregate alveolar surfactant (H) to a light-aggregate,nonsurface active form (L) is believed to involve the activity of anenzyme, namely, convertase. This conversion can bereproduced in vitro by the surface-area cycling technique. The purposeof the present study was to use this technique to investigate thedevelopmental aspects of convertase activity in fetal, newborn, andadult rabbits. H was isolated from alveolar lavage from term[31-day gestation (31d)] fetal rabbit pups, 1-, 4-, and7-day-old newborns, and adults, and the percent conversion to L wasdetermined. To assess lamellar bodies (LB) as a potential source ofactivity in this species, these structures were isolated from lungtissue of 27-day-gestation (27d) and 31d fetuses, 1-, 4-, and 7-day-oldnewborns, and adults and were cycled the same as for H. LB containedconsiderable activity at each developmental stage i.e., ~82% of a27d LB preparation converted to L after 3 h of cycling. In the adult,this value was 78%. Very little conversion of H was obtained fromfetal lung (i.e., <20% of the 31d fetal preparation converted to L),but, by postnatal day 4, this valuewas greatly increased (i.e., >80% conversion) and stayed elevated toadulthood. The activity for each H and LB fraction was temperature andconcentration dependent and diminished with storage at 4°C. Thesedata suggest the LB as the source of convertase activity in the rabbitand demonstrate dramatic developmental changes in this activity afterrelease of the LB contents to the alveoli.

     

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.     

Differences in airway structure in immature and mature rabbits.

Our laboratory has previously demonstrated that maximal bronchoconstriction produces a greater degree of airway narrowing in immature than in mature rabbit lungs (33). To determine whether these maturational differences could be related to airway structure, we compared the fraction of the airway wall occupied by airway smooth muscle (ASM) and cartilage, the proportion of wall area internal to ASM, and the number of alveolar attachments to the airways, from mature and immature (6-mo- and 4-wk-old, respectively) rabbit lungs that were formalin fixed at total lung capacity. The results demonstrate that the airway walls of immature rabbits had a greater percentage of smooth muscle, a lower percentage of cartilage, and fewer alveolar attachments compared with mature rabbit airways; however, we did not find maturational differences in the airway wall thickness relative to airway size. We conclude that structural differences in the airway wall may contribute to the greater airway narrowing observed in immature rabbits during bronchoconstriction.     

Size distribution of recruited alveolar volumes in airway reopening.

In 11 isolated dog lung lobes, we studied the size distribution of recruited alveolar volumes that become available for gas exchange during inflation from the collapsed state. Three catheters were wedged into 2-mm-diameter airways at total lung capacity. Small-amplitude pseudorandom pressure oscillations between 1 and 47 Hz were led into the catheters, and the input impedances of the regions subtended by the catheters were continuously recorded using a wave tube technique during inflation from -5 cm H(2)O transpulmonary pressure to total lung capacity. The impedance data were fit with a model to obtain regional tissue elastance (Eti) as a function of inflation. First, Eti was high and decreased in discrete jumps as more groups of alveoli were recruited. By assuming that the number of opened alveoli is inversely proportional to Eti, we calculated from the jumps in Eti the distribution of the discrete increments in the number of opened alveoli. This distribution was in good agreement with model simulations in which airways open in cascade or avalanches. Implications for mechanical ventilation may be found in these results.     

Changes in surfactant pools after a physiological increase in alveolar surfactant

We have used previously characterized models to investigate the reuptake of surfactant from the alveolus. In model 1, rats were swum in a water bath at 33 degrees C for 30 min, which increased tidal volume (VT) approximately 300% and frequency 60%; they were then allowed to rest for up to 4 h. In model 2, rats were exposed to 5% CO2-13% O2-82% N2 for 24 h, which increased both VT and frequency approximately 200%; these rats were then rested for up to 24 h. In both models we harvested a tissue fraction (lamellar bodies, lb) and two alveolar fractions--tubular myelin rich (alv-1) and tubular myelin poor (alv-2). Immediately after swimming, lb-dipalmitoylphosphatidylcholine (DPPClb) was 18% below the control of 0.94 +/- 0.037 (SE) mg/g wet lung (n = 24 rats; P less than 0.05); this returned to control by 2 h. Whereas DPPCalv-1 was constant at all time points, DPPCalv-2 was increased 50% above the control of 2.68 +/- 0.085 mg/g dry lung (n = 27 rats; P less than 0.001) immediately and up to 1 h after swimming. It returned to control levels between 2 and 3 h. After gas exposure, DPPC in lb, alv-1, and alv-2 was 33, 64, and 89%, respectively, above controls. All three fractions had normalized after 24 h. Our results demonstrate marked differences in the response of the surfactant system to acute and more prolonged stimuli. Of particular interest was the constancy of alv-1 with swimming, suggesting that it may be the controlled variable. However, the system appeared to be reset by prolonged hyperpnea, a process that may involve an increase in synthesis of surfactant.     

Clearance of 99mTc-DTPA and experimentally increased alveolar surfactant content.

We measured clearance of 99mTc-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA) in rabbits with experimentally increased alveolar surfactant content. In one group of animals, surfactant production was increased by treatment with ambroxol, and another group of animals was treated with tracheal instillation of natural surfactant. A group of untreated control animals and animals treated with instillation of saline were also studied. Clearance was measured during standard conditions of mechanical ventilation and during ventilation with large tidal volumes. In ambroxol- and surfactant-treated groups, clearance rate was reduced compared with untreated control animals. In contrast, clearance rate increased after saline instillation. The differences were observed at both modes of ventilation. The findings indicate that the pulmonary surfactant system is a rate-limiting factor for the clearance of 99mTc-DTPA and that the volume dependence of clearance is not explained by stretching of the alveolar wall only.     

Different ventilation strategies alter surfactant responses in preterm rabbits.

The effect of ventilation strategy on in vivo function of different surfactants was evaluated in preterm rabbits delivered at 27 days gestational age and ventilated with either 0 cmH2O positive end-expiratory pressure (PEEP) at tidal volumes of 10-11 ml/kg or 3 cmH2O PEEP at tidal volumes of 7-8 ml/kg after treatment with one of four different surfactants: sheep surfactant, the lipids of sheep surfactant stripped of protein (LH-20 lipid), Exosurf, and Survanta. The use of 3 cmH2O PEEP decreased pneumothoraces in all groups except for the sheep surfactant group where pneumothoraces increased (P < 0.01). Ventilatory pressures (peak pressures - PEEP) decreased more with the 3 cmH2O PEEP, low-tidal-volume ventilation strategy for Exosurf-, Survanta-, and sheep surfactant-treated rabbits (P < 0.05), whereas ventilation efficiency indexes (VEI) improved only for Survanta- and sheep surfactant-treated rabbits with 3 cmH2O PEEP (P < 0.01). Pressure-volume curves for sheep surfactant-treated rabbits were better than for all other treated groups (P < 0.01), although Exosurf and Survanta increased lung volumes above those in control rabbits (P < 0.05). The recovery of intravascular radiolabeled albumin in the lungs and alveolar washes was used as an indicator of pulmonary edema. Only Survanta and sheep surfactant decreased protein leaks in the absence of PEEP, whereas all treatments decreased labeled albumin recoveries when 3 cmH2O PEEP was used (P < 0.05). These experiments demonstrate that ventilation style will alter a number of measurements of surfactant function, and the effects differ for different surfactants.