Pulmonary dysfunction in neonatal SP-B-deficient mice

Keisuke Tokieda1, Jeffrey A. Whitsett1, Jean C. Clark1, Timothy E. Weaver1, Kazushige Ikeda1, Keith B. McConnell1, Alan H. Jobe2, Machiko Ikegami2, and Harriet S. Iwamoto1

1 Divisions of Neonatology and Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039; and 2 Department of Pediatrics, Harbor-University of California Los Angeles Medical Center, Torrance, California 90509

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pulmonary function was assessed in newborn wild-type and homozygous and heterozygous surfactant protein B (SP-B)-deficient mice after birth. SP-B+/+ and SP-B+/- mice became well oxygenated and survived postnatally. Although lung compliance was decreased slightly in the SP-B+/- mice, lung volumes and compliances were decreased markedly in homozygous SP-B-/- mice. They died rapidly after birth, failing to inflate their lungs or oxygenate. SP-B proprotein was absent in the SP-B-/- mice and was reduced in the SP-B+/- mice, as assessed by Western analysis. Surfactant protein A, surfactant proprotein C, surfactant protein D, and surfactant phospholipid content in lungs from SP-B+/- and SP-B-/- mice were not altered. Lung saturated phosphatidylcholine and precursor incorporation into saturated phosphatidylcholine were not influenced by SP-B genotype. Intratracheal administration of perfluorocarbon resulted in lung expansion, oxygenation, and prolonged survival of SP-B-/- mice and in reduced lung compliance in SP-B+/+ and SP-B+/- mice. Lack of SP-B caused respiratory failure at birth, and decreased SP-B protein was associated with reduced lung compliance. These findings demonstrate the critical role of SP-B in perinatal adaptation to air breathing.

lung function; perfluorocarbon; surfactant protein; respiratory distress syndrome; lung compliance

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SURFACTANT PROTEIN (SP) B is a 79-amino acid amphipathic polypeptide associated with surfactant phospholipids in the alveolus of the lung (6). SP-B is encoded by a single gene located on chromosome 2 in humans and at a syntenic site on chromosome 6 in the mouse (15). The SP-B gene encodes a 40- to 42-kDa glycosylated preproprotein that is proteolytically processed by type II epithelial cells to produce the active peptide detected within the alveolus (22). The recent observations that the genetic ablation of murine SP-B causes lethal respiratory failure at birth and that mutations in the human SP-B gene cause lethal respiratory distress in full-term newborn infants provide strong support for the critical role of SP-B in lung function (4, 17). SP-B binds phospholipids, causes lipid fusion, and contributes to the formation of multilayer lipid membranes. SP-B also plays a critical role in the intracellular processing of surfactant, being required for the formation of lamellar bodies and the proteolytic processing of surfactant proprotein C (proSP-C). The addition of SP-B and other surfactant proteins, SP-A and SP-C, to phospholipid mixtures enhances adsorption and development of low surface tension at an air-liquid interface (Ref. 9, for review see Ref. 23).

The complete lack of SP-B in newborn mice and humans causes lethal respiratory failure at birth (4, 17, 18). Infants with hereditary SP-B deficiency can be temporarily supported by mechanical ventilation, extracorporeal membrane oxygenation, and exogenous surfactant replacement (8). Lethal respiratory failure in these infants has been corrected only by lung transplantation. Partial defects in SP-B can also cause respiratory failure in full-term infants, and decreased amounts of SP-B occur in preterm infants with respiratory distress syndrome (19) and in adults with acute respiratory distress syndrome (7). Developmental, humoral, inflammatory, and spatial regulatory factors influence SP-B gene expression (23). Because inactivation of a single SP-B allele decreased SP-B protein in adult SP-B+/- mice (3), the present study was designed to evaluate aspects of lung mechanics, lung function, and phospholipid metabolism in newborn heterozygous and homozygous SP-B-deficient mice. Furthermore, because there are no effective pharmacological therapies for SP-B deficiency in human neonates, the present study also evaluated the effects of perfluorocarbon on survival and lung function in newborn SP-B-deficient mice.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ablation of the murine SP-B gene by insertion of the neomycin resistance gene into the fourth exon of the murine gene was accomplished in embryonic stem cells (D3), as previously described (4). Newborn pups were obtained by mating SP-B+/- parents.

Cesarean section and resuscitation. Pregnant mice were anesthetized (2-3% isoflurane) at 18-19 days of gestation, and pups were delivered within 3 min. Dams were then killed by exsanguination. After delivery, pups were placed in an incubator maintained at 33-35°C with an inspired O2 fraction of >40% and were observed for at least 30 min after delivery. Pups were monitored for breathing activity and skin color. A clinical score based on breathing activity and skin color was recorded every 10 min for 30 min [clinical score = breathing activity + skin color; breathing activity was scored as 0 (none), 1 (gasping), or 2 (continuous breathing); skin color was scored as 0 (blue), 1 (dusky), or 2 (pink)]. One group of pups (20 SP-B+/+, 23 SP-B+/-, and 12 SP-B-/-) received no treatment. A second group of pups (10 SP-B+/+, 15 SP-B+/-, and 13 SP-B-/-) received perfluorocarbon (FC-75, 3 M) immediately after delivery. Perfluorocarbon (50 µl) was administered into the oropharynx with a pipette. The respiratory efforts of the mice facilitated delivery of perfluorocarbon to the lungs.

Pressure-volume analysis. Breathing mice were killed with an overdose of pentobarbital sodium (200 mg/kg ip) and were placed in 100% O2 to permit the complete collapse of the alveolar space by O2 absorption. The trachea was cannulated transorally (24-gauge Angiocath; Becton-Dickinson, Rutherford, NJ) and was connected to a syringe and a pressure sensor (X-ducer; Motorola, Phoenix, AZ) via a three-way connecter. Air leaks around the catheter were prevented by application of glue (Quick tight; Loctite) around the catheter. Lungs were inflated in 10-µl increments to a maximum inflation pressure of ~20 cmH2O and were deflated to negative pressure. Pressure and volume on inflation and deflation were recorded and were normalized to the weight of the animal. Lung compliance was determined from the slope of the deflation curve, where the curve approximated a straight line between -10 and +10 cmH2O. The hysteresis ratio was calculated as the area bound by the inflation and deflation curves divided by the total area bound by the maximum and minimum values for pressure and volume area using software kindly provided by Dr. Gary S. Huvard (Huvard Research and Consulting, Chesterfield, VA). Similar measurements were made in SP-B-/- mice that failed to inflate their lungs within 40 min after delivery. These animals had severe respiratory failure (low clinical scores at 30 min). Lung compliance measurements were completed by 40-50 min after delivery in these animals. In animals without respiratory failure (high clinical scores at 30 min), lung compliance measurements were performed within 2-3 h after delivery.

Histology. Lungs from neonatal SP-B+/+, SP-B+/-, and SP-B-/- mice were fixed after passive deflation by immersion in 4% paraformaldehyde, embedded in paraffin, cut into 5-µm sections, and stained with hematoxylin and eosin. A total of 32 lungs were studied histologically. All lobes of each lung were examined at two different levels (~25 µm apart). Nine SP-B+/+, thirteen SP-B+/-, and ten SP-B-/- mice were studied. At least three animals of each genotype were exposed to room air, O2, or O2 + perfluorocarbon for 30 min before euthanasia. Lung sections were examined and were scored by three individuals who did not know the source of the histological samples. Epithelial, vascular, and alveolar portions of the lungs were scored on the basis of 0% (0), <= 50% (1), <= 75% (2), or 100% (3) evidence of damage or presence of alveolar exudate. These scores were averaged and analyzed by nested analysis of variance and contrast comparisons.

Genotyping. DNA was isolated from tail or liver tissue. Genotypes were determined using polymerase chain reaction amplification, as described previously (3). Briefly, oligonucleotide primers specific for the neomycin resistance gene and intron 4 of the SP-B gene were used to identify a 1.3-kb product from the mutated allele. Oligonucleotide primers to introns 3 and 4 of the SP-B gene identified a 260-base pair product from the wild-type SP-B allele. Mutated and wild-type alleles were assessed in all mice.

SP-B protein analysis. For Western blot analysis, fetal lung tissues were collected on day 18 of gestation. Tissue was homogenized in 10 mM tris(hydroxymethyl)aminomethane (Tris, pH 7.5), 0.25 M sucrose, 1 mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of pepstatin A, aprotinin, antipain, leupeptin, and chymostatin, and the supernatant was centrifuged at 140 g for 10 min (4°C). Equal amounts of protein from the supernatant were dissolved in electrophoresis sample buffer and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic transfer to nitrocellulose membrane, as previously described (4). The membrane was blocked with 5% milk in Tris-buffered saline and was incubated with rabbit anti-human SP-B antibody overnight at room temperature. Subsequently, the membrane was washed with Tris-buffered saline containing 0.05% Tween 20 and 0.05% Nonidet P-40, incubated with a 1:20,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Calbiochem, La Jolla, CA) for 2 h at room temperature, washed again, and developed with a chemiluminescent substrate [enhanced chemiluminescence (ECL); Amersham, Arlington Heights, IL].

Lung saturated phosphatidylcholine. On day 17 of gestation, three pregnant mice were injected intraperitoneally with 0.5 ml of saline containing 0.5 mCi of [3H]palmitic acid ([9,10-3H]palmitic acid, 60 Ci/mmol; American Radiochemicals, St. Louis, MO) and 42 µCi of [methyl-14C]choline chloride (40 Ci/mol; New England Nuclear, Boston MA). Mice were sedated with an intraperitoneal injection of pentobarbital sodium 6 h later, the fetuses were delivered and chilled on ice, and the livers and lungs were removed and homogenized. The lipids were extracted with 2:1 chloroform-methanol. Phosphatidylcholine was isolated from the liver extract by one-dimensional thin-layer chromatography, and equivalent spots were used for quantification of radioactivity and phosphorus (10). Specific activity, defined as radioactivity divided by micromoles of phosphatidylcholine, was calculated for each fetal liver. Saturated phosphatidylcholine (Sat PC) was isolated from the lung extract after treatment with OsO4 by aluminum column chromatography, according to Mason et al. (13). Specific activities for Sat PC from the fetal lung were calculated from radioactivity and phosphorus measurements. Incorporation of the radiolabels into liver phosphatidylcholine was used to estimate isotope transfer to each fetus.


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Fig. 1.   Radiolabeling of lung saturated phosphatidylcholine (Sat PC) relative to liver phosphatidylcholine in fetal mice. Specific activities (SA) for lung Sat PC and liver phosphatidylcholine (cpm/µmol) were normalized by dividing SA of lung Sat PC by SA of liver phosphatidylcholine for fetuses in a litter and then dividing by mean ratio for litter. Normalized SA values are grouped by surfactant protein B (SP-B) genotype [+/+ (n = 5), +/- (n = 19), -/- (n = 9)]. Mean values for groups are not different across groups for [3H]palmitate (F = 0.24, P = 0.80) or [14C]choline (F = 0.58, P = 0.56).

Statistical analysis. To compare specific activities for Sat PC among litters and among genotypes, the ratio of specific activities of lung Sat PC to liver phosphatidylcholine was calculated for each mouse. The mean of the ratio for all mice in each litter was calculated, and the ratio for each mouse was expressed relative to the mean. The normalized ratios for SP-B+/+, SP-B+/-, and SP-B-/- mice were grouped, and the group mean ± SE was calculated for each radiolabeled precursor. Differences in other variables among the three genotypes (SP-B+/+, SP-B+/-, and SP-B-/-) were assessed by nested or two-way analysis of variance, and differences between means were assessed by contrast comparisons and the Student-Newman-Keuls test.


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Fig. 2.   Clinical scores for respiratory failure in untreated (A) and perfluorocarbon-treated (B) newborn mice. All mice were observed for at least 30 min after birth. Breathing activity and skin color were assessed at 10, 20, and 30 min. Clinical scores of SP-B-/- mice without treatment at 20 min (P < 0.01) and 30 min (P < 0.001) are significantly lower than those of SP-B+/+ and SP-B+/- mice without treatment. Clinical score of SP-B-/- mice after perfluorocarbon treatment at 30 min significantly improved (P < 0.05) compared with that of SP-B-/- mice without treatment at 30 min but was lower (P < 0.01) than that of SP-B+/+ mice after perfluorocarbon treatment at 30 min. * Significantly different from SP-B+/+ and SP-B+/-. dagger  Significantly different from SP-B -/-, without treatment, 30-min value.


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Fig. 3.   Pressure-volume curves of lungs from untreated SP-B+/+ (A), untreated SP-B+/- (B), untreated SP-B-/- (C), perfluorocarbon-treated SP-B+/+ (D), perfluorocarbon-treated SP-B+/- (E), and perfluorocarbon-treated SP-B-/- (F) mice. Inflation-deflation curves were generated from newborn mice at 18 days of gestation.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Surfactant proteins. Genotyping of the SP-B+/+, SP-B+/-, and SP-B-/- fetal mice demonstrated the expected frequency of inheritance of the SP-B allele as an autosomal recessive gene. As previously described for full-term newborn mice (4), SP-B protein, assessed by Western analysis of lung tissue homogenates, was not detected in newborn homozygous mice (data not shown). Lung SP-B protein was reduced in SP-B+/- compared with wild-type littermates, whereas SP-A, proSP-C, and SP-D were not altered in the SP-B+/- or SP-B-/- mice. The proSP-C fragment (10-12 kDa) and decreased SP-C active peptide (4 kDa) were observed in SP-B-/- pups but not in SP-B+/+ or SP-B+/- neonates. The abnormal proSP-C fragment characteristic of SP-B-/- mice was also detected by Western blot analysis of amniotic fluid but was not detected in amniotic fluid from SP-B+/+ or SP-B+/- mice (data not shown).


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Fig. 4.   Lung measurements in SP-B+/+, SP-B+/-, and SP-B-/- mice and effects of perfluorocarbon. Pressure-volume curves were generated in untreated (n = 20) and perfluorocarbon-treated (n = 10) SP-B+/+, untreated (n = 23) and perfluorocarbon-treated (n = 15) SP-B+/-, and untreated (n = 12) and perfluorocarbon-treated (n = 13) SP-B-/- mice. Lung volume at 18 cmH2O inflation pressure, lung compliance, and hysteresis ratio were significantly reduced in SP-B-/- mice compared with SP-B+/+ mice (P < 0.001). Only lung compliance in SP-B+/- mice was significantly reduced compared with SP-B+/+ mice (P < 0.05). After treatment with perfluorocarbon, most values were significantly changed when values of same genotype with no treatment were compared. Lung volumes, compliances, and hysteresis ratios were not significantly different among genotypes after perfluorocarbon treatment. * Significantly different from SP-B+/+. dagger  Significantly different from same genotype in untreated group. Values are means ± SE. For details on how these values were generated see MATERIALS AND METHODS.


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Fig. 5.   Perfluorocarbon treatment improved lung inflation in SP-B-/- mice. Lungs were obtained from SP-B-/- mice that were exposed to room air (A), 40% O2 (B), or perfluorocarbon and 40% O2 (C) for 30 min after cesarean delivery. Lungs obtained from mice exposed to room air or O2 alone were collapsed and showed no evidence of gas exchange. In contrast, lungs obtained from SP-B-/- mice exposed to perfluorocarbon and O2 showed evidence of lung expansion and gas exchange. Sections (5 µm) were stained with hematoxylin and eosin. Scale bar, 100 µm.

Lung Sat PC. In mice from three separate litters, there were similar amounts of Sat PC in the lungs of SP-B+/+, SP-B+/-, and SP-B-/- mice: 82.5 ± 8.5 (n = 5), 75.3 ± 3.5 (n = 19), and 66.4 ± 4.7 µmol/kg (n = 9), respectively (F = 1.8, P = 0.2 by analysis of variance). There were no differences in lung Sat PC among the genotypes. The trend toward lower lung Sat PC amounts for the SP-B-/- mice is explained by more mice being from the litter with the lowest amounts of lung Sat PC than from the other litters. Incorporation of the radiolabeled precursors [3H]palmitic acid and [14C]choline into lung Sat PC also was not different for the mice, differing only in SP-B genotype (Fig. 1).


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Fig. 6.   Perfluorocarbon treatment produced evidence of lung damage. Lungs were obtained from SP-B-/- mice that were exposed to 40% O2 (A) or perfluorocarbon and 40% O2 (B-D) for at least 2 h. O2 exposure alone had no significant effect on bronchiolar epithelium (b) or arteriolar structure (a). B: perfluorocarbon treatment markedly disrupted bronchiolar epithelium. Areas of flattened or absent epithelial cells were common. C: arteriolar structure was disrupted, as evidenced by areas where endothelial layer has pulled away from the rest of vessel structure. D: exudates composed of proteinaceous material were abundant in alveolar structures. Cells and cellular debris were also commonly present. Histological structure of SP-B+/+ and SP-B+/- mice treated in the same manner were similar. Sections (5 µm) were stained with hematoxylin and eosin. Scale bar, 22 µm.

Clinical features after delivery. Most of the SP-B+/+ and SP-B+/- mice survived postnatally (survival rates were 97.7 and 94%, respectively) and were oxygenated within 20 min in association with visible lung inflation. In contrast, all SP-B-/- mice died of respiratory failure with poor clinical scores (Fig. 2) within 30 min after birth, despite vigorous respiratory efforts. Perfluorocarbon was easily delivered by the oropharyngeal route. All mice survived for at least 30 min after perfluorocarbon treatment. The clinical scores of SP-B-/- mice at 30 min after perfluorocarbon treatment were significantly improved (P < 0.05) compared with those of untreated SP-B-/- mice (Fig. 2). Although SP-B-/- mice were rescued by perfluorocarbon, the clinical improvement did not achieve normal clinical scores characteristic of SP-B+/+ or SP-B+/- mice. Lungs of SP-B-/- mice receiving perfluorocarbon became visually inflated, but unlike SP-B+/+ and SP-B+/- mice, lungs of the SP-B-/- mice virtually collapsed at the end of expiration.

Pulmonary function and morphology. Pressure-volume curves of SP-B-/- mice were typical of surfactant deficiency, with decreased compliance, no hysteresis, and low residual and total lung volumes (Figs. 3 and 4). Data from individual animals are shown in Fig. 3, and group data are shown in Fig. 4. Lung volumes at 18 cmH2O pressure were markedly decreased in SP-B-/- mice (Figs. 3 and 4). Although SP-B+/- mice successfully initiated ventilation, lung compliance was significantly, although modestly, reduced in SP-B+/- mice compared with the SP-B+/+ littermates (Fig. 4). Consistent with pressure-volume analysis, lungs of the SP-B-/- mice remained atelectatic with low hysteresis ratios (Fig. 4), whereas lungs of SP-B+/- and SP-B+/+ mice were inflated and indistinguishable from each other at the light-microscopic level (Fig. 5). Perfluorocarbon treatment markedly improved the pressure-volume curve of SP-B-/- mice, restoring lung volume and compliance and increasing the hysteresis ratio (Fig. 4). In contrast, perfluorocarbon significantly decreased the lung compliance of SP-B+/+ and SP-B+/- mice (Fig. 4). Pressure-volume curves, lung compliance, and hysteresis after perfluorocarbon treatment were not significantly different among SP-B+/+, SP-B+/-, and SP-B-/- mice (Figs. 3 and 4).

Histological examination of lungs obtained from SP-B+/+, SP-B+/-, or SP-B-/- mice exposed to room air, 40% O2, or 40% O2 and perfluorocarbon demonstrated that O2 exposure alone for 30 min had no effect, whereas the combination of O2 and perfluorocarbon treatment significantly altered lung structure. Lung histology of the SP-B-/- mice demonstrated improvement in inflation after perfluorocarbon treatment, consistent with the ability of the perfluorocarbon to reduce surface tension in the air spaces (Fig. 5). Evidence of damage to epithelial, vascular, and alveolar structures was observed after perfluorocarbon treatment in SP-B-/- mice after 30 min and in all groups after exposure for longer times (Fig. 6). Damage after 30 min of perfluorocarbon treatment was most severe in the SP-B-/- mice (Fig. 7).


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Fig. 7.   Summary of histological effects of 30-min exposure to room air, O2, or O2 and perfluorocarbon treatment. Scores of appearance of bronchial epithelial (A), vascular (B), and alveolar structures (C) are shown for SP-B+/+, SP-B+/-, and SP-B-/- neonatal mice. Values are means ± SE of an average of scores recorded by 3 individuals on 3-5 samples/group. * Significantly different from same genotype exposed to room air or O2 (P < 0.0001). § Significantly different from animals with different genotypes also exposed to perfluorocarbon (P < 0.001).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mutation of the SP-B gene by homologous recombination completely ablated SP-B mRNA and protein in homozygous transgenic mice and reduced the levels of SP-B mRNA and protein in heterozygous SP-B+/- mice (Refs. 3 and 4 and present study). In the present study, absence of SP-B caused lethal respiratory failure that was ameliorated by the administration of perfluorocarbon, which enhanced oxygenation, increased lung volume and compliance, and increased the time of survival. Although heterozygous SP-B-deficient mice survived cesarean delivery, lung compliance was decreased slightly compared with wild-type littermates, supporting the concept that normal amounts of SP-B are required for normal perinatal lung function. The finding that decreased SP-B is associated with pulmonary dysfunction in SP-B+/- and SP-B-/- newborn mice is consistent with the increased lung function and compliance observed after treatment of surfactant-deficient preterm animals with exogenous surfactant containing SP-B (9). Although SP-B is subject to a variety of temporal and humoral influences in the neonatal period, gene dosage appears to play an important role in the control of steady-state SP-B production. Whereas SP-B peptide was completely absent in the SP-B-/- newborn mice and was reduced in the SP-B+/- newborn mice, the amount of lung Sat PC and the incorporation of choline and palmitate into Sat PC were not influenced by the presence or level of SP-B.

SP-B is essential for the formation of lamellar bodies within type II cells and for the normal processing of SP-C (4, 18). Reduction of SP-B in heterozygous mice is apparently sufficient to result in normal lamellar body formation and proSP-C processing in newborn or adult SP-B+/- mice. Complete reduction of SP-B by gene knockout surprisingly does not affect lung Sat PC pool sizes and precursor incorporation by type II cells. Because of the small size of the newborn pups, we were unable to assess Sat PC or other lipids in bronchoalveolar lavage fluid. The location, processing, and fate of Sat PC is unknown but apparently is not affected by the absence of SP-B protein. Normal incorporation of radiolabeled precursors into phosphatidylcholine in lung tissue explants from an infant with SP-B deficiency was recently reported (2).

Reduction in SP-B content in the lungs of neonatal SP-B+/- mice results in a modest reduction in lung compliance without changes in lung volume. The reduction in total lung SP-B content in newborn SP-B+/- mice was comparable to that observed in adult SP-B+/- mice and was consistent with an ~50% decrease in SP-B content in bronchoalveolar lavage previously observed in the adult SP-B+/- mice (3). The small decrease in lung compliance in the neonatal SP-B+/- mice was similar to that recently observed in adult SP-B+/- mice (3). However, other differences between SP-B+/+ and SP-B+/- mice, such as the air trapping consistently observed in the adult SP-B+/- mice, were not apparent in the neonatal animals. Whether the difference in air trapping in neonatal compared with adult SP-B+/- mice is related to developmental differences in local levels of SP-B expression in the respiratory epithelium or to intrinsic anatomic or mechanical differences between neonatal and adult mouse lungs is unclear.

Pulmonary function in the SP-B-/- mice was markedly impaired with decreased lung volume and compliance typical of surfactant dysfunction seen in premature infants with severe respiratory distress syndrome. Despite gasping activity, administration of O2, and vigorous stimulation, none of the SP-B-/- mice survived beyond 30 min of age. The acute disruption in lung function observed in the SP-B-/- mice is apparently due to the absence of SP-B protein and not to the presence of an aberrant form of SP-C propeptide. Recent cross-breeding studies demonstrated that SP-B-/- mice survive to adulthood if they express the NH2-terminal portion of human SP-B in their lungs (1). These transgenic mice produce SP-B and the aberrant form of SP-C propeptide and survive normally.

Administration of perfluorocarbon in the immediate postnatal period was associated with prolongation of survival, improved oxygenation, and visual inflation of the lung of SP-B-/- mice. Pressure-volume curves demonstrated markedly improved compliance and increased lung volumes after perfluorocarbon treatment of the SP-B-/- mice. Perfluorocarbon also altered lung function and mechanics in SP-B+/+ and SP-B+/- mice, likely related to its intrinsic surface tension-lowering properties, viscosity, and ablating effects of endogenous surfactant on lung function (21). All lung compliance curves, lung volumes, and hysteresis ratios were similar after perfluorocarbon treatment, indicating that differences among genotypes were not related to tissue structure. Breathing patterns in the treated SP-B-/- mice were distinct from those in the SP-B+/+ and SP-B+/- mice: the lungs of SP-B-/- mice collapsed at end expiration. These findings suggest that the minimum surface tension of perfluorocarbon is not sufficient to maintain complete alveolar stability at end expiration. In the surfactant-sufficient lung, minimum surface tension of surfactant is believed to be <1 dyn/cm2 and is much lower than that of the perfluorocarbon (FC-75), which is ~15 dyn/cm2 (11). Whereas perfluorocarbon improved lung function, the treatment caused histological abnormalities of the lung, affecting the bronchial and bronchiolar epithelium, vascular structures, and presence of alveolar exudate. Histological abnormalities were most severe in SP-B-/- mice and were not observed in untreated animals of any genotype. Whether histological abnormalities resulted from perfluorocarbon or lack of endogenous surfactant is unclear.

In the present study, modest reductions in SP-B were associated with modest changes in pulmonary mechanics in newborn SP-B+/- mice. Because the levels of SP-B are reduced in premature infants and SP-B expression is decreased in association with lung injury, infection, and O2 exposure (23), decreased SP-B may play a role in the pathogenesis of clinical lung disease. Likewise, human heterozygous SP-B gene carriers may be susceptible to pulmonary dysfunction at birth, especially if born prematurely.

Perfluorocarbon dramatically improved lung function in SP-B-/- mice, similar to findings in premature animals and infants (12). This surfactant-like activity of perfluorocarbon has been described when administered as partial liquid ventilation (5, 12, 14, 20, 21). Long-term survival of homozygous SP-B-deficient patients requires lung transplantation. Because exogenous surfactant therapy has not been successful in the treatment of neonatal SP-B deficiency in humans (8), the present findings support the concept that perfluorocarbon may be useful in stabilizing critically ill SP-B-/- infants during the early phase of their therapy. The cytopathology observed after perfluorocarbon treatment suggests that such therapy may not be successful for long-term therapy of hereditary SP-B deficiency.

    ACKNOWLEDGEMENTS

We thank Dr. Susan E. Wert for assistance with the histological analyses. We acknowledge the assistance of Ann C. Maher, the technical assistance of Sherri A. Profitt with histology, and the help of Dr. Mark Kurtzman and Jennifer Melzer with anesthesia.

    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-51832.

Address for reprint requests: H. S. Iwamoto, Div. of Neonatology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039.

Received 2 July 1996; accepted in final form 17 June 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(4):L875-L882
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