Natural and artificial lung surfactant replacement therapy in premature lambs

The effect of tracheal instillation of surface-active mixtures in premature lambs was studied as an animal model of exogenous surfactant replacement therapy for the respiratory distress syndrome (RDS). Specific mixtures studied were 7:3 (molar ratio) dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) and extracted mixed lipids (with 1% protein) from cow lung lavage (CLL). Preventilatory tracheal instillation of greater than 15 mg/kg of CLL in 10 ml 0.15 M NaCl to premature lambs gave improved alveolar-arterial O2 gradient and blood gases and increased lung compliance, compared with control lambs over a 15-h period. Lambs receiving 7:3 DPPC:PG dispersions were not improved over controls with regard to pressure-volume characteristics and were worse than controls in arterial oxygenation. In terms of in vitro surface properties, both extracted natural CLL and 7:3 DPPC:egg PG were able to lower aqueous surface tension to 1 dyn/cm under dynamic compression. However, the dynamic respreading of CLL films on successive surface cycles was superior to that of 7:3 DPPC:PG. Moreover, after dispersal in 0.15 M NaCl by vortexing (5 mg/80 ml), CLL adsorbed to surface pressure (tau values of 45 dyn/cm within 10 min. 7:3 DPPC:PG adsorbed to significantly lower tau values after subphase dispersal by a variety of methods.     

Aerosolized surfactant treatment of preterm lambs

To evaluate the potential for aerosolized surfactant treatments of surfactant deficiency, twin lamb fetuses were delivered at 130-132 days gestational age and received nebulized natural surfactant (Neb NS), nebulized Survanta (Neb Surv), tracheally instilled natural surfactant (Inst NS), or nebulized saline (Neb Saline). Neb NS and Neb Surv groups had significant increases in ventilatory efficiency index and dynamic compliance values (P less than 0.05). Both groups also had pressure-volume curves that were comparable to the Inst NS group. The Neb Saline control group had deterioration of the ventilation efficiency index and dynamic compliance values over time as well as pressure-volume curves that demonstrated smaller lung volumes compared with all three surfactant-treated groups (P less than 0.01). Delivery of aerosolized surfactant to the lung was only approximately 2 mg lipid/kg for the nebulized groups, a dose one-twentieth of that previously noted to be effective in instillation protocols. Distribution histograms of the aerosolized surfactant-treated groups differed from the instilled animals as there was more deposition in the right upper lobes and tracheae in the nebulized groups compared with the instilled group (P less than 0.05). Pulmonary blood flow was not altered by aerosolized surfactant treatment. Administration of aerosolized surfactant to preterm lambs improved lung function at a very low surfactant dose.     

Surface activity of the surfactant in experimental compression atelectasis

Surface surfactant activity was studied at different time periods of compression atelectasis, induced by hydrothorax in 35 guinea pigs. The animals were slaughtered 30 and 60 minutes or 3, 12 and 24 hours after hydrothorax. It has been demonstrated that experimental compression atelectasis is accompanied by surface activity lowering, associated with disorders in surfactant secretion into the alveolar lumen. The qualitative composition of surfactant phospholipids remains unchanged, which may play an essential role in the recovery of lung tissue aeration.     

Surface activity in vitro: role of surfactant proteins

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.     

Surfactant inhibition by plasma: gestational age and surfactant treatment effects in preterm lambs

Ikegami, Machiko, Celso M. Rebello, and Alan H. Jobe.Surfactant inhibition by plasma: gestational age and surfactant treatment effects in preterm lambs. J. Appl.Physiol. 81(6): 2517-2522, 1996.The preterminfant with respiratory distress syndrome has edematous lungs and smallamounts of surfactant that do not function normally. We reported thatsurfactant recovered from preterm lambs after surfactant treatment canhave decreased sensitivity to inhibition of surface tension by plasma.We asked whether this augmented resistance to inhibition was dependenton lung development (gestational age) by testing sensitivity to plasmainhibition of 1) endogenous surfactant from preterm lambs and 2)surfactant from preterm lambs after treatment with an organicsolvent-extracted natural sheep surfactant. Surfactant recovered aftersurfactant treatment of 121- or 128-days-gestation lambs had the samesensitivity to plasma inhibition as did the surfactant used to treatthe lambs. Surfactant recovered from 134-days-gestation lambs haddecreased sensitivity to inhibition. Lung maturation is a variableinfluencing surfactant inhibition by plasma.

     

Tidal volume effects on surfactant treatment responses with the initiation of ventilation in preterm lambs

Wada, Kazuko, Alan H. Jobe, and Machiko Ikegami. Tidalvolume effects on surfactant treatment responses with the initiation ofventilation in preterm lambs. J. Appl.Physiol. 83(4): 1054-1061, 1997.We hypothesizedthat initiation of ventilation in preterm lambs with high volumes wouldcause lung injury and decrease the subsequent response to surfactanttreatment. Preterm lambs were randomized to ventilation for 30 minafter birth with 5 ml/kg (VT5),10 ml/kg (VT10), or 20 ml/kg(VT20) tidal volumes and then ventilated with ~10 ml/kg tidal volumes to achieve arterialPCO₂ values of ~50 Torr to 6 h ofage. VT20 lambs had lowercompliances, lower ventilatory efficiencies, higher recoveries ofprotein, and lower recoveries of surfactant in alveolar lavages and in surfactant that had decreased compliances when tested in preterm rabbits than VT5 orVT10 lambs. Other lambsrandomized to treatment with surfactant at birth and ventilation with6, 12, or 20 ml/kg tidal volumes for 30 min had no indicators of lunginjury. An initial tidal volume of 20 ml/kg decreased the subsequentresponse to surfactant treatment, an effect that was prevented withsurfactant treatment at birth.

     

Growth factor concentrations and growth-promoting activity in human milk following premature birth

We have measured growth factor concentrations in human milk from mothers of term and premature infants to identify any adaptive responses to premature delivery. Measurements included concentrations of epidermal growth factor and insulin and the growth-promoting activity of milk in vitro, estimated by the stimulation of rats of protein accumulation in cultured human fibroblasts. Compared with women delivering at full-term, mothers of premature infants produced milk containing higher concentrations of epidermal growth factor and increased growth-promoting activity in vitro, changes which were probably maintained throughout lactation. The anabolic effect of human milk in cultured human fibroblasts could be attributed partially but not entirely to epidermal growth factor, suggesting that the concentrations of additional growth factors were also increased following premature delivery. Insulin did not contribute to the extra growth-promoting activity; premature delivery depressed the insulin concentration significantly on the first two days of lactation and, thereafter, milk from mothers of term or premature infants contained similar amounts of insulin. Growth factor concentrations were also measured in cow's milk-based formulae. These formulae contained low concentrations of epidermal growth factor and insulin and reduced growth-promoting activity compared with human milk. Changes in milk growth factor concentrations may occur as a compensatory mechanism to accelerate growth and development in pre-term infants, and if so, it follows that premature infants could benefit more from their own mother's milk than from pooled human milk or from cow's milk-based formulae.     

Pulmonary effects of acute prenatal asphyxia in ventilated premature lambs

The effect of profound repetitive prenatal asphyxial insults on the cardiopulmonary function of premature ventilated lambs was studied. Twenty-nine fetal lambs (approximately 138 days gestational age) were exteriorized. In 16 of these lambs, the umbilical cord was occluded for 4 min then released for 10 min. This asphyxial episode was repeated until the arterial pH was approximately 7.00, and the mean arterial blood pressure was less than 40 mmHg and falling. The 13 control lambs were simply exteriorized with the umbilical circulation intact. The lambs were then ventilated for 3-4 h. There were no differences between the control vs. asphyxiated lambs in pulmonary compliances (0.57 and 0.58 ml.cmH2O-1.kg-1) wet-to-dry weight ratios (8.18 and 7.55), cardiac outputs (177.8 and 141.8 ml.kg-1.min-1), surfactant-saturated phosphatidylcholine pool sizes, or atrial and/or ductal shunts. Asphyxia did not interfere with the redirection of blood away from atelectatic lung segments created by bronchial obstruction with balloon catheters. Also, although the bidirectional flux of protein into and out of the airways of these preterm lambs was large relative to term lambs, there was no effect of asphyxia on this protein leak. In this animal model, prenatal asphyxia did not impact negatively on the severity of the respiratory failure.     

Microbial activity in soils following steam treatment

Steam enhanced extraction (SEE) is an aquifer remediation technique that can be effective at removing the bulk of non-aqueous phase liquid (NAPL) contamination from the subsurface, particularly highly volatile contaminants. However, low volatility compounds such as polynuclear aromatic hydrocarbons (PAHs) are less efficientlyremoved by this process. This research evaluated the effects of steam injection on soilmicrobial activity, community structure, and the potential for biodegradation of contaminants following steam treatment. Three different soils were evaluated: alaboratory-prepared microbially-enriched soil, soil from a creosote contaminated fieldsite, and soil from a chlorinated solvent and waste oil contaminated field site. Results from field-scale steaming are also presented. Microbial activity before and after steam treatment was evaluated using direct epifluorescent microscopy (DEM) using the respiratory activity dye 5-cyano-2,3, ditolyl tetrazolium chloride (CTC) in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) to yield a quantitative assessment of active and total microbial numbers. DEM results indicate that steamed soils that were analyzed while still hot exhibited microbial activity levels that were below detection. However, soil samples that were slowly cooled, more closely reflecting the conditions of applied SEE, exhibited microbial activity levels that were comparable to presteamed soils. Samples from a field-site where steam was applied continuously for 6 weeks also showed high levels of microbial activity following cooling. The metabolic capabilities of the steamed communities were investigated by measuring cell growth in enrichment cultures on various substrates. These studies provided evidence that organisms capable of biodegradation were among the mesophilic populations that survived steam treatment. Fluorescent in situ hybridization (FISH) analysis of the soils with domain-level rRNA probes suggest that both Archaea and Bacteria survived steam exposure.     

Respiratory effects of a patent ductus arteriosus in premature newborn lambs

We examined the respiratory effects of a patent ductus arteriosus in 29 premature lambs (131-135 days gestational age) after infiltrating the ductal wall with formaldehyde solution (Formalin) and placing a snare around the ductus to regulate its patency. The lambs were given sheep surfactant, paralyzed, and mechanically ventilated at birth. We first compared 8 lambs with open ductus and 13 lambs with closed ductus during the 12 h after birth. Although lambs with open ductus had greater pulmonary blood flow (301 +/- 36 vs. 188 +/- 11 ml.min-1.kg-1, mean +/- SE, at 12 h of age) and mean pulmonary arterial (44 +/- 3 vs. 33 +/- 2 mmHg) and left ventricular end-diastolic (6 +/- 0.6 vs 4 +/- 0.7 mmHg) pressures, we found no differences in dynamic respiratory compliance (Cdyn = 0.55 +/- 0.07 vs. 0.55 +/- 0.03 ml.cmH2O-1.kg-1), midtidal volume resistance (62 +/- 5 X 10(-3) vs. 62 +/- 7 X 10(-3) cmH2O.ml-1.s), or functional residual capacity (FRC = 27 +/- 3 vs. 26 +/- 2 ml.kg-1). Alveolar-arterial PO2 difference was lower in the lambs with open ductus (238 +/- 65 vs. 362 +/- 37 Torr). Next, we challenged eight lambs with two separate saline infusions (50 ml.kg-1 over 3 min), each given with the ductus alternately closed or open. When the ductus was closed, FRC was unchanged, but Cdyn increased by 18% immediately after the infusion. When the ductus was open, FRC decreased by 16% and Cdyn decreased by 12%. We conclude that the premature lamb is surprisingly resistant to changes in respiratory function from ductal patency during the immediate neonatal period.     

A study of following behaviour in young lambs

The following behaviour of young domestic lambs was studied by observing their responses to a surrogate ewe moving around a circular runway. Lambs from 12 h to 3 weeks of age were tested once individually. In one series of 67 5-min tests made outdoors, some lambs followed the surrogate throughout while others did not follow at all. The median following time was 42 s. In a second series, consisting of 34 15-min indoor tests, the median following time of the lambs was greater (450 s, 50% of test time), probably because this was a more stressful environment. In both series of tests, lambs of 6 to 10 days of age followed the surrogate more than younger or older lambs. Following behaviour did not appear to change when the recorded bleats of each lamb's dam were replayed instead of the recorded bleat of an alien ewe. It was concluded that management practices which increase the opportunity for very young lambs, especially twin lambs, to contact and follow alien ewes need careful control to avoid separation of dam and off-spring.     

The labelling of pulmonary surfactant phosphatidylcholine in 19-31-days-old lambs

The labelling of surfactant phosphatidylcholine and disaturated phosphatidylcholine was studied in 19-31-days-old lambs. Following the placement of small bore tracheal catheters, the animals were given radioactively labelled palmitic acid and/or choline by intravenous injection and multiple samples were recovered from the distal airways of each animal via a small catheter. The specific activities of the phosphatidylcholine and/or disaturated phosphatidylcholine were measured in these samples of surfactant. The labelled phospholipids accumulated in the samples of surfactant in a linear fashion; the mean time required to reach maximal specific activities in phosphatidylcholine and saturated phosphatidylcholine with either palmitic acid or choline as precursor was 28 h. Subsequently the specific activities of the labelled phospholipids from the surfactant samples decreased semi-logarithmically. The mean t1/2 for phosphatidylcholine and disaturated phosphatidylcholine labelled with radioactive palmitic acid was 35 h. The saturated phosphatidylcholine labelled with radioactive choline had a t1/2 of 251 h. The results demonstrate that surfactant labelling studies can be done by multiple sampling of single large animals.