Intrapulmonary catabolism of surfactant-saturated phosphatidylcholine in rabbits.

Intrapulmonary surfactant catabolism was investigated by use of a phospholipase A1- and A2-resistant analogue of dipalmitoylphosphatidylcholine (DPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPC ether). [14C]DPC ether, made into liposomes with [3H]DPC and associated with 32P-labeled rabbit surfactant, was given intratracheally to 1-kg rabbits, which were killed at preset times to 48 h. Recoveries of radiolabel as saturated phosphatidylcholine (Sat PC) isolated from alveolar wash (AW), postlavage lung homogenate (LH), and alveolar macrophages were measured. All groups had similar AW and LH Sat PC pool sizes, indicating no perturbation of endogenous Sat PC pools. Despite a nearly fivefold accumulation of [14C]DPC ether in the lung by 48 h (P less than 0.01), the three probes had similar alveolar clearance curves. Furthermore, the Sat PC reutilization efficiency (41.6%) and turnover time (5.9 h) calculated for DPC ether were not different from values for the DPC and rabbit surfactant. Of the DPC ether (0.7%) and DPC (9%) labels recovered as PC in organs outside the lung, greater than 85% was unsaturated, indicating de novo synthesis using precursors from degraded PC. DPC ether was a useful probe of intrapulmonary DPC catabolism, and after alveolar uptake there was no direct reentry of intact DPC from the catabolic compartment(s) into the secretory pathway.

Distribution of surfactant and ventilation in surfactant-treated preterm lambs.

Surfactant-deficient ventilated preterm lambs were treated with 100 mg/kg of surfactant radiolabeled with microspheres at 30 min and 2.5 h of age to evaluate the effect of treatment technique on surfactant distribution. The treatments were four positions with four boluses (bolus 4), two lateral positions with two boluses (bolus 2), or a 30-min infusion (infusion). The bolus groups had uniform surfactant distributions to the > 100 pieces analyzed for each lung. Infusion resulted in a very nonuniform surfactant distribution (P < 0.01). Surfactant was recovered equivalently in all lobes of the bolus groups, whereas infusion lungs contained surfactant preferentially in upper lobes (P < 0.01). The second dose of surfactant localized into the same lung doses as the first dose (P < 0.001). Blood flow increased proportionately to surfactant content in the bolus groups. With infusion, blood flow decreased and ventilation measured with 99Tc-labeled aerosol increased to pieces of lung receiving large amounts of the infusion surfactant, suggesting that localized overinflation was likely. Physiological measurements indicated better responses to bolus treatments, although the infusion lambs did improve. These results indicate that different treatment techniques can have large effects on surfactant distributions.

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.

Antenatal betamethasone dose effects in preterm rabbits studied at 27 days gestation.

Pregnant rabbits received bethamethasone (0.05, 0.2, 0.4, or 0.5 mg.kg-1.day-1) or vehicle control for 2 days before delivery of fetuses at 27 days gestation to evaluate dose-related effects on surfactant pool sizes with and without ventilation, pressure-volume measurements, lung protein leaks, and precursor incorporation into lung saturated phosphatidylcholine (PC). Alveolar wash-saturated PC pool sizes in betamethasone-exposed fetuses were less than in controls (P less than 0.01). At higher doses, total lung saturated PC also decreased (P less than 0.01). Maximal lung volumes on pressure-volume loops were larger than controls only at the 0.4 mg.kg-1.day-1 dose (P less than 0.05). The larger maximal volumes, despite decreased saturated PC pools, indicated increased responsiveness of the steroid-treated lungs to the smaller saturated PC pool sizes. Vascular-to-alveolar iodinated albumin leak decreased with steroid treatment independently of dose (P less than 0.01). No consistent pattern of increased precursor incorporation into saturated PC by lung slices was seen. Our results indicate that, in preterm rabbits exposed to a range of maternal corticosteroid doses, the beneficial lung maturational effect of structural alterations with increased responses to endogenous saturated PC pools was maximal even at the lowest dose.

Lung parenchyma and type II cell morphometrics: effect of surfactant treatment on preterm ventilated lamb lungs.

The effect of exogenous surfactant treatment on lung and type II cell structure of ventilated lambs of 137-138 days gestational age was studied. Thirty-four lambs were delivered and randomized to control or 100 mg/kg of natural sheep surfactant treatment groups. Lungs from one group of lambs not treated with surfactant were fixed before ventilation, and the other animals were ventilated to maintain normal blood gas values for 3, 24, or 48 h. Morphometric assessment of the inflation-fixed lung parenchyma of ventilated lungs was compared with the architectural appearance of alveoli and alveolar ducts in the unventilated lungs. Mechanical ventilation resulted in distension of alveolar ducts accompanied by the shallowing and loss of well-defined alveoli and areas of atelectasis at 3 h. These abnormalities increased in severity after 24 and 48 h of ventilation. Surfactant treatment before ventilation significantly reduced the extent and degree of dilatation and concomitant atelectasis. The fraction of normal parenchyma was 38 +/- 7% in untreated lambs vs. 64 +/- 6% in treated lambs after 24 h of ventilation. After 48 h of ventilation, significant differences between control (39 +/- 6%) and surfactant-treated (55 +/- 6%) lambs were still evident. Alveolar type II cells contained approximately 15% lamellar bodies by volume. Neither surfactant treatment nor time of ventilation altered the volume density of lamellar bodies or other organelles, except for a decrease in glycogen from 8% in nonventilated lungs to 2.5% in lungs ventilated for 24 h. These findings indicate that a surfactant treatment at birth results in the maintenance of more normal parenchyma with less atelectasis during prolonged ventilation of the immature lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of rabbit lung lysosomes and their role in surfactant dipalmitoylphosphatidylcholine catabolism.

Although alveolar surfactant is rapidly catabolized in adult rabbit lungs, the pathways have not been characterized. Pathways of surfactant secretion and recycling involve lamellar bodies and multivesicular bodies, organelles shown to be related to lysosomes by cytochemistry and autoradiography. Since lysosomes are central to intracellular catabolic events, it is possible that lysosomes are involved in intrapulmonary surfactant catabolism. Lysosomes relatively free of contaminating organelles (as determined morphologically and by marker enzymes for mitochondria, endoplasmic reticulum, peroxisomes, and plasma membranes) were obtained from post-lavage lung homogenates of 1-kg rabbits by differential centrifugation in buffered sucrose and gradient separation in percoll (density, 1.075-1.165). The role of lung lysosomes in catabolism of dipalmitoylphosphatidylcholine (DPC) was then studied in rabbits killed 4, 12, and 24 h following intratracheal injection of [3H]DPC and [14C] dihexadecyl phosphatidylcholine (DPC-ether). While equal amounts of label were in the lamellar body containing fractions at 4 h, nearly 6-fold more DPC-ether label than DPC label was recovered in the lysosomal fractions. By 24 h, there was 15-fold more DPC-ether in the lysosomes. This is the first report of successful isolation of lysosomes relatively free of other organelles from rabbit lungs. The tracer studies indicate DPC and DPC-ether follow similar intracellular processing after alveolar uptake. The subsequent accumulation of the ether analog in the lysosomal fractions supports a role for these organelles in surfactant DPC catabolism.

Bacteremic infection with Pediococcus: vancomycin-resistant opportunist.

Pediococci are recently recognized Gram-positive human pathogens, resistant to vancomycin and generally susceptible to penicillin. Infection in adults has been seen in patients with chronic underlying conditions as well as those with previous abdominal surgery. Two previous infants with congenital gastrointestinal malformations requiring surgical correction have been reported with sepsis attributable to Pediococcus sp. We report a third infant born with gastroschisis who developed Pediococcus bacteremia and meningitis 3 months after surgery, and speculate regarding the role of probiotics in the pathogenesis of this infection.

Localization of alveolar surfactant clearance in rabbit lung cells.

Localization of surfactant phospholipid clearance in lung cells was investigated in vivo in rabbits using radiolabeled dipalmitoylphosphatidylcholine (DPPC) and 1,2-dihexa-decyl-sn-glycero-3-phosphocholine (DPPC-ether), a phospholipase A1- and A2-resistant analogue of DPPC. After intratracheal injection of liposomes of the labeled lipids associated with unlabeled surfactant, adult rabbits were killed in groups of three to five at 0, 4, 12, and 24 h with recovery of bronchoalveolar lavages for alveolar macrophages and surfactant. Type II cells and tissue-associated macrophages were isolated on Percoll gradients following elastase and trypsin digestion of the lungs. Radiolabel recoveries as saturated phosphatidylcholine were measured in alveolar wash, alveolar macrophages, lung tissue, and the type II cell and mixed cell bands from the Percoll gradients. Cost accounting of label demonstrated similar recoveries at 0 h, but significantly more DPPC-ether compared with DPPC in cells at later times, indicating ineffective degradation of the DPPC-ether. Internalization of the lung tissue-associated labels into cells was time dependent. At all times, greater than 65% of the cell-associated labels were recovered in type II cells, indicating the primary role for these cells in clearing alveolar surfactant phospholipid in vivo. The total contribution of alveolar macrophages to the overall clearance was approximately 20%.

Dose effects of antenatal corticosteroids for induction of lung maturation in preterm rabbits.

Dose-response effects of corticosteroids were investigated in the preterm rabbit. Pregnant rabbits were given two doses of 0.01, 0.03, or 0.1 mg of betamethasone per kilogram every 24 hours, beginning on day 25 of gestation. Saline solution was used in a comparison treatment group. Half of the newborn rabbits received supplemental surfactant therapy after delivery via cesarean section on day 27, and all were ventilated on a ventilator-plethysmograph system for 30 minutes. The 0.01 and 0.03 mg/kg regimens had no effect on birth weight or lung function. The 0.1 mg/kg regimen resulted in fetal growth retardation and improved ventilatory measurements, gas exchange, and decreased protein accumulation in the lung, without increasing surfactant pool size. The induction of lung maturation by corticosteroids has multiple targets in the developing lung and does not exhibit a linear dose response.

Lysosomes from rabbit type II cells catabolize surfactant lipids.

The role of a lysosome fraction from rabbit type II cells in surfactant dipalmitoylphosphatidylcholine (DPPC) catabolism was investigated in vivo using radiolabeled DPPC and dihexadecylphosphatidylcholine (1, 2-dihexadecyl-sn-glycero-3-phosphocholine; DEPC), a phospholipase A(1)- and A(2)-resistant analog of DPPC. Freshly isolated type II cells were gently disrupted by shearing, and lysosomes were isolated with Percoll density gradients (density range 1.0591-1.1457 g/ml). The lysosome fractions were relatively free of contaminating organelles as determined by electron microscopy and organelle marker enzymes. After intratracheal injection of rabbits with [(3)H]DPPC and [(14)C]DEPC associated with a trace amount of natural rabbit surfactant, the degradation-resistant DEPC accumulated 16-fold compared with DPPC in lysosome fractions at 15 h. Lysosomes can be isolated from freshly isolated type II cells, and lysosomes from type II cells are the primary catabolic organelle for alveolar surfactant DPPC following reuptake by type II cells in vivo.

Surfactant metabolism during hyperventilation of newborn lambs with atrial right to left shunts.

We studied the effect on surfactant metabolism of 8 h of mechanical ventilation at tidal volumes of 13 +/- 0.3 ml/kg and very high tidal volumes of 28 +/- 1.5 ml/kg, with and without added CO2, in the presence of an atrial right to left shunt in 4- to 8-day-old lambs. Similarly aged, spontaneously breathing lambs were used as controls. Right to left atrial shunts were created by inflating a balloon in the right atrium after a Rashkind atrial septostomy, thus creating a stable, easily controlled atrial shunt. Radiolabeled surfactant phospholipid precursors were used to probe incorporation into and secretion of surfactant phosphatidylcholine, whereas intratracheally administered labeled natural surfactant was used to evaluate alveolar clearance. Protein leak from the vascular space to the lungs was measured using radioactive iodine-labeled albumins. At the end of the 8-h study period, tissue association of alveolar surfactant was significantly increased to 63% in the mechanically hyperventilated lambs as compared to 44% in those lambs mechanically ventilated but not hyperventilated (p less than 0.05) and to 39% in the spontaneously breathing control animals (p less than 0.05). No increased surfactant secretion or decreased compliance was detected with hyperventilation. However, the lambs had very large surfactant-saturated phosphatidylcholine pool sizes, and a large portion (50%) was already in the alveolar pool, even in the spontaneously breathing lambs. Precursor incorporation into saturated phosphatidylcholine was similar in all groups, and very low and comparable protein leaks were seen in the different groups of lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung albumin recovery in surfactant-treated preterm ventilated lambs.

Preterm ventilated animals and infants with respiratory distress syndrome (RDS) develop proteinaceous alveolar edema. To study the effect of postnatal age on intravascular radiolabeled albumin accumulation into lungs, preterm lambs at 132 days gestational age were ventilated after treatment with sheep surfactant or cow surfactant extract for periods as long as 24 h. Lambs not treated with surfactant were studied for only 5 h because of severe respiratory failure. All lambs were given radiolabeled albumin by intravascular injection 1 h before they were killed, and the net recovery of the labeled albumin was measured in the lung tissue and air space as quantified by alveolar lavage. Net 1-h radiolabeled albumin recoveries in the lungs decreased from 5 to 6% soon after birth to 0.9% at 24 h in the surfactant-treated groups (p less than 0.01). At 3 h there was less labeled albumin recovery by alveolar lavages in lambs treated with sheep surfactant than in control lambs and lambs treated with cow surfactant extract (p less than 0.05). Protein in alveolar washes from lambs treated with cow surfactant extract exceeded that in lambs treated with sheep surfactant at 3 h (p less than 0.05), but protein recoveries had decreased to similar values by 24 h, indicating a net clearance of air-space protein. These studies demonstrate a sixfold decrease in net albumin accumulation from birth to 24 h of age despite continued ventilation and oxygen exposure of the premature lamb lungs.

Surfactant protein A metabolism in preterm ventilated lambs.

Surfactant protein A (SP-A) metabolism was studied in vivo in 33 preterm ventilated lambs at 138 +/- 1 days gestational age by measuring recoveries of exogenously administered surfactant containing both radiolabeled SP-A and labeled saturated phosphatidylcholine (Sat PC) given via the trachea at birth. Endogenously secreted SP-A was also labeled with [35S]methionine and followed over 24 h. The exogenously labeled SP-A left the alveolar pool more rapidly than did Sat PC over the first 5 h of life (P less than 0.05), and both exogenously labeled SP-A and Sat PC were detected within lamellar bodies by 2 h, indicating uptake from the airspaces. The quantity of SP-A in alveolar washes increased about twofold from birth to 5 h of age, whereas alveolar Sat PC pools were constant over 24 h. The SP-A endogenously labeled with [35S]methionine was recovered at highest specific activities in the alveolar washes at 10 and 45 min after birth with no labeled SP-A detectable in lamellar body fractions until 2 h. The curve for endogenous SP-A labeling of lamellar bodies was similar to that for exogenous labeling, indicating that SP-A was initially secreted by a pathway independent of lamellar bodies with subsequent SP-A labeling of lamellar bodies. The kinetics of SP-A metabolism were very different than for Sat PC in preterm lambs.

Treatment responses to surfactants containing natural surfactant proteins in preterm rabbits.

The in vivo function of surfactants reconstituted using natural surfactant lipid and protein constituents was evaluated in 27-day-gestation preterm rabbits. The animals were treated with protein-free surfactant lipids (LH-20), LH-20 + 5% SP-A, LH-20 + 1% SP-B, LH-20 + 1% SP-C, LH-20 + 5% SP-A + 1% SP-B + 1% SP-C (SP-ABC), natural sheep surfactant, or 4 ml/kg 0.45% NaCl (control) and then ventilated with tidal volumes of 8 ml/kg and 3 cm H2O positive end-expiratory pressure (PEEP). Ventilatory pressures (peak pressures minus PEEP) and dynamic compliances of the LH-20 + SP-C rabbits were greater (p < 0.01) than those of control, LH-20, and LH-20 + SP-A groups but lower (p < 0.05) than in the LH-20 + SP-B, LH-20 + SP-ABC, and sheep surfactant groups. Recoveries of intravascular labeled albumin in the lungs were comparable in the LH-20 + SP-B, LH-20 + SP-C, LH-20 + SP-ABC, and sheep surfactant groups and less (p < 0.01) than in LH-20 + SP-A rabbits, which had lower (p < 0.05) recoveries than did the control and LH-20 groups. The postventilation pressure-volume curves for LH-20 + SP-B and LH-20 + SP-ABC rabbits had significantly lower opening pressures, larger maximal lung volumes, and larger retained volumes on deflation relative to the LH-20 + SP-C, LH-20, and control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased surfactant dose-response after delayed administration to preterm rabbits.

Preterm rabbits from 14 litters were delivered at 27 d gestation, anesthetized, and treated with surfactant at birth, 15 min, or 30 min after the onset of mechanical ventilation. Doses of surfactant ranging from 0 to 100 mg/kg body weight were given intratracheally and the rabbits were ventilated for 45 min after birth. Pressure-volume curves and dynamic compliances demonstrated that the dose response to surfactant progressively decreased with delayed treatment. Following surfactant treatments of 50 mg/kg at birth, 15 min, and 30 min, peak lung volumes at 35 cm H2O were increased by 49, 30, and 8.4%, respectively over those of untreated controls. Lung lavages from rabbits receiving surfactant at 30 min had significantly higher protein contents and minimum surface tensions on the Wilhelmy balance than those from rabbits treated at birth (23.1 +/- 1.1 versus 16.0 +/- 2.7 dynes/cm), and lung sections from rabbits treated at 30 min had a significantly less uniform distribution of surfactant than those from rabbits treated at birth. While increasing phospholipid concentrations may reverse the inhibition of surfactant by serum proteins in vitro, there was a progressive inability of exogenous surfactant to overcome this inhibition in vivo following delayed administration to very immature rabbits. This inability to overcome inhibition with increasing surfactant dose was associated with a less uniform distribution of surfactant following its delayed administration.

In vivo function of surfactants containing phosphatidylcholine analogs.

Increased phospholipase A2 activity demonstrated in some forms of lung injury may contribute to surfactant dysfunction. Phospholipase A2-resistant analogs of dipalmitoylphosphatidylcholine (DPPC) with surfactant properties might therefore be useful lipid components of treatment surfactants for certain lung injuries. The in vivo function of surfactants containing DPPC or the phospholipase-resistant analogs dihexadecylphosphatidylcholine (DEPC) or dihexadecylphosphonotidylcholine (DEPnC), with or without surfactant proteins B and C (SP-B+C), was thus evaluated in preterm rabbits (27 days' gestation). Rabbits randomly received one of seven surfactants (DPPC, DEPC, DEPnC, DPPC+SP-B+C, DEPC+SP-B+C, DEPnC+SP-B+C, or lipid extract surfactant [LES]) or 0.45% NaCl (control) and were ventilated for 30 min. Lipid-only surfactants decreased ventilatory pressures (peak inspiratory pressures minus positive end-expiratory pressure) relative to control (p < 0.05). Addition of SP-B+C further decreased ventilatory pressures to levels similar to LES (p < 0.01 versus control, lipid-only surfactants). Lung dynamic compliances and postventilation pressure-volume curves improved in the following order: LES, SP-B+C lipid surfactants > lipid-only surfactants > control (p < 0.05). All surfactant preparations decreased intravascular 125I-albumin recoveries in the lungs relative to control (p < 0.01 for all surfactants versus control). These results indicate that DEPC and DEPnC were as effective as DPPC as lipid components of synthetic surfactants. And like DPPC, the analogs interacted with isolated SP-B+C and improved in vivo function to levels comparable to LES.

A free radical scavenger (Lazaroid U75412E) attenuates tumor necrosis factor-alpha generation in a rabbit model of smoke-induced lung injury.

The lazaroid (21-aminosteroid) analogue U75412E was evaluated in rabbits exposed to diesel fuel-polycarbonate plastic smoke. Inhalation of total of 4.6 mg U75412E aerosolized at a rate of 1.53 mg/min for 3 min before or after smoke significantly prevented or limited the extent of alveolar hypoventilation, interstitial edema, and tumor necrosis factor-alpha (TNF-alpha) by pulmonary alveolar macrophages (PAM) ex vivo observed at 2 h. The smoke-induced changes in wet lung/body weight ratios and the production of superoxide (O2-) by PAM ex vivo were also attenuated by the drug treatment after smoke exposure (p < 0.05). This study suggests that lazaroids may ameliorate the oxygen-radical-initiated cytokine processes and inflammation cascade as a result of the smoke insult.