- Email: [email protected]
Extremely preterm fetal sheep lung responses to antenatal steroids and inflammation
Accepted Manuscript Extremely preterm fetal sheep lung responses to antenatal steroids and inflammation Kevin Visconti, MD, Paranthaman Senthamaraikannan, PhD, Matthew W. Kemp, PhD, Masatoshi Saito, MD PhD, Boris W. Kramer, MD PhD, John P. Newnham, MD, Alan H. Jobe, MD PhD, Suhas G. Kallapur, MD PII:
S0002-9378(17)32698-4
DOI:
10.1016/j.ajog.2017.12.207
Reference:
YMOB 11995
To appear in:
American Journal of Obstetrics and Gynecology
Received Date: 23 June 2017 Revised Date:
27 November 2017
Accepted Date: 14 December 2017
Please cite this article as: Visconti K, Senthamaraikannan P, Kemp MW, Saito M, Kramer BW, Newnham JP, Jobe AH, Kallapur SG, Extremely preterm fetal sheep lung responses to antenatal steroids and inflammation, American Journal of Obstetrics and Gynecology (2018), doi: 10.1016/ j.ajog.2017.12.207. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Extremely preterm fetal sheep lung responses to antenatal steroids
Kevin Visconti MD
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and inflammation
Paranthaman Senthamaraikannan PhD
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Matthew W. Kemp PhD Masatoshi Saito MD PhD
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Boris W. Kramer MD PhD John P. Newnham MD Alan H. Jobe MD PhD
Suhas G. Kallapur MD Disclosure: The authors report no conflict of interest
Contact information:
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Suhas G. Kallapur MD Professor of Pediatrics
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Funding: Financial Markets Foundation for Children Grant to MWK, MS and JPN.
Neonatal Research Center of the UCLA Children’s Discovery and Innovation Institute, Division of Neonatology & Developmental Biology, Department of Pediatrics, David
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Geffen School of Medicine UCLA. Mattel Children's Hospital UCLA 10833 Le Conte Avenue, Room B2-375 MDCC Los Angeles, CA 90095 Tel: 310-206-8489
Fax: 310-267-0154 Email id: [email protected] Word count: 2980
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Condensation: Fetal sheep lungs comparable to peri-viable human lungs have decreased maturational or inflammatory responses compared to more mature preterm
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sheep lungs.
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Short-version of the title: Effect of the antenatal steroid in the periviable fetal lung
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Glossary of terms:
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ATP1B1 - Sodium potassium ATPase (Na-K ATPase) (Ion channel) ANS - Antenatal steroids Beta - Betamethasone CAT - Catalase (antioxidant enzyme) CCTα - Phosphocholine cytidyltransferase a (an intermediary in the surfactant lipid pathway) CDP-choline Cytidine diphosphate choline (an intermediary in the surfactant lipid pathway) CEPT1 - Choline/Ethanolamine phoshphotransferase 1 (an intermediary in the surfactant lipid pathway) CTGF - Connective tissue growth factor CRP - C-reactive protein (an acute phase reactant) CTP - Cytidine triphosphate CYR61 - Cysteine rich angiogenic inducer 61 (an acute phase reactant) DPPC - dipalmitoylphosphatidylcholine (component of surfactant lipid) EGR1- Early growth response protein-1 FASN - Fatty acid synthase (enzyme in the surfactant lipid synthesis pathway) GPX1 - Glutathione peroxidase (antioxidant enzyme) IL1beta - Interleukin 1 beta IL6 - Interleukin 6 IL8 - Interleukin 8 LPCAT1- Lysophosphatidylcholine acyltransferase (enzyme in the surfactant lipid synthesis pathway) LPS - lipopolysaccharide MCP1 - Monocyte chemoattractant protein 1 MPO - Myeloperoxidase (used as a neutrophil marker) Na-K ATPase - Sodium Potassium ATPase (ion channel) NOS - Nitric oxide synthase NR3C - Nuclear Receptor Subfamily 3 Group C (steroid receptor) NR4A - Nuclear Receptor Subfamily 4 Group A Member 1 PC - Phosphatidyl choline (intermediary in surfactant lipid) PU.1 - A transcription factor that is expressed in maturing monocytes/macrophages RT-PCR - Reverse transcriptase polymerase chain reaction Pro-SPC - A precursor protein of surfactant protein C SCNN1- Epithelial sodium channel (formerly called eNac) SOD - Superoxide dismutase (antioxidant enzyme) SP-A - Surfactant protein A SP-B - Surfactant protein B SP-C - Surfactant protein C SP-D - Surfactant protein D TNFa - Tumor necrosis factor alpha TTF-1 - Thyroid transcription factor 1 (a marker of alveolar type II cell) VEGF - Vascular endothelial growth factor
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ABSTRACT 244 words Background: The efficacy of antenatal steroids for fetal lung maturation in the peri-
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viable period is not fully understood.
Objective(s): To determine the lung maturational effects of ANS and inflammation in early gestation sheep fetuses, similar to the peri-viable period in humans.
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Study Design: Date-mated ewes with singleton fetuses were randomly assigned to one of 4 treatment groups (n=8/group): 1) maternal intramuscular injection (IM) of
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betamethasone (Beta), 2) intra-amniotic lipopolysaccharide (IA) (LPS), 3) Beta+LPS, 4) IA+IM Saline (controls). Fetuses were delivered surgically 48h later at 94d gestation (63% term gestation) for comprehensive evaluations of lung maturation, lung and systemic inflammation.
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Results: Relative to controls, 1) Betamethasone increased the fetal lung air-space tomesenchymal area ratio by 47% but did not increase the mRNAs for the surfactant
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proteins (SP) B and C that are important for surfactant function or increase the expression of pro-SP-C in the alveolar type II cells. 2) Betamethasone increased
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expression of 1 of the 4 genes in surfactant lipid synthetic pathways, 3) Betamethasone increased genes involved in epithelium sodium channel transport, but not Na-K ATPase or Aquaporin 5. 4) Lipopolysaccharide increased pro-inflammatory genes in the lung but did not effectively recruit activated inflammatory cells. 5) Betamethasone incompletely suppressed Lipopolysaccharide induced lung inflammation. In the liver, Betamethasone when given alone increased the expression of serum amyloid A3 and C-reactive protein mRNAs.
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Conclusion(s): Compared the more mature 125d gestation sheep, antenatal steroids do not induce pulmonary surfactants during the peri-viable period, indicating a different
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response.
Key words: fetal lung maturation, chorioamnionitis, surfactant, respiratory distress
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syndrome, fetal inflammation
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INTRODUCTION Antenatal steroids (ANS) for at risk pregnancies from 24 to 34 weeks gestational age are the standard of care since the NIH consensus statement of 1995.1 However,
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only about 100 women treated with ANS delivered before 28 weeks in all of the single randomized trials performed before 1993.2 Thus, there are limited high quality data to support benefit of ANS at early gestation despite increasing populations of these
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infants.3 Several large observational studies recently demonstrated efficacy of ANS at the margins of viability.4-8 The ACOG/SMFM Obstetrical Clinical Consensus (October
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2017) states that antenatal steroids are not recommended prior to 23w0d, may be "considered" at 23w0d, and are recommended for 24w0d gestation and beyond 9. However, there may be an inherent selection bias in outcomes in the observational studies since pregnancies in the peri-viable period given ANS likely receive more
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survival directed care.
Although early gestation human lung explants and fetal lung cells in culture respond to corticosteroids with morphologic changes and induction of surfactant
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components,10 whether these changes occur in vivo is not known. ANS increase lung volumes in fetal monkeys at the margin of viability, but the effects on surfactant system
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are not known.11 Thus, the question of response characterisitics and efficacy of ANS at the margins of viability is not fully settled. Fetal exposure to chorioamnioitis can decrease the incidence of respiratory
distress syndrome by inducing early lung maturation clinically,
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a phenomenon also
demonstrated in animal models.13 In fetal sheep models, the combined fetal exposure to inflammation and corticosteroids induce more lung maturation than either stimulus
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alone.14 However, either exposure may have adverse fetal effects: fetal inflammatory responses with fetal injury for inflammation and decreased fetal growth and concerns about neurodevelopment for corticosteroids 15, 16.
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Although there are some studies at earlier gestational ages, most of the randomized clinical data and experimental data in sheep for effects of antenatal steroids are for more than 80% term gestation.2,17, 18 Despite their common occurences, there is
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lack of information regarding early gestation effects of combined exposures to antenatal steroids and inflammation on the fetal lung 5, 19. We hypothesized that, in early gestation
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fetal sheep, both ANS and intra-uterine inflammation would increase indicators of lung
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maturation and the two exposures would further increase the maturational signals.
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METHODS Study Design With the approval of Animal Ethics Committee at The University of Western
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Australia, time-mated ewes with singleton fetuses were randomly assigned to one of 4 treatment groups: 1) Intra-amniotic injection of lipopolysaccharide (LPS) (10 mg Escherichia coli 055:B5, Sigma Chemical, St. Louis, MO), 2) Maternal intramuscular
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injection of betamethasone 0.5 mg/kg maternal weight [Celestone Soluspan, ScheringPlough, New South Wales, Australia], 3) Intra-amniotic LPS and betamethasone in
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combination, or 4) Intra-amniotic + intra-muscular injections of saline (controls) at 92 days GA (n=8 animals/group). This betamethasone dose and duration of exposure of 2d was based on previous studies demonstrating increases in surfactant protein-B and surfactant protein-C mRNA in Beta exposed 124d gestation sheep
20, 21
, but the
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experimental dose is higher than what is used clinically. The maternal betamethasone was given about 3 h prior to the intra-amniotic injection of LPS
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to allow time for fetal
exposure of Beta prior to IA LPS. This experimental design reported previously
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allowed comparison of effects in the 124d gestation to the 94d gestation sheep. Some animals were used for fetal a pulmonary vascular study 23.
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Animal sampling protocol
Lambs were surgically delivered 2d after exposures at 94d GA and euthanized
with 100 mg/kg pentobarbital. Each fetus was weighed and fetal cord blood was collected for cell counts. The lungs were removed, separated, and weighed. Lung tissue from the right lower lobe was snap frozen and the right upper lobe was inflation fixed in 10% buffered formalin at 30 cm H2O pressure for 24 h. The very immature late
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canalicular lung structure will not support ventilation for physiologic measurements of lung mechanics. Messenger RNA Quantification
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Total RNA extraction from frozen tissue samples and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed as previously described to quantify expression of key genes involved in surfactant function, lung fluid
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homeostasis, and lung inflammation. 24 Immunohistochemistry
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Immunohistochemistry assessed the distribution of alveolar type II cells and inflammatory cells in the fetal lung
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.
Briefly, the sections were incubated with
antibodies against Myeloperoxidase (Cell Marque, Rocklin, CA, cat # 289A-75, dilution 1:200), PU.1 (Santa Cruz Biotechnology, Santa Cruz, CA, cat # sc-352, dilution 1:500)
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TTF-1 (Seven Hills, Cincinnati, OH, cat # WMAb--8G7G31, dilution 1:500), Pro-SPC (Seven Hills, Cincinnati, OH, cat # R460, dilution 1:500) in 2% serum at 4oC overnight. Immunostaining was visualized using a Vectastain ABC peroxidase kit (Vector
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Laboratories Inc., Burlingame, CA). Slides were counterstained with Nuclear Fast Red for photomicroscopy.
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Lung Morphometry and alveolar type II cell counts To determine air-space fraction, images of lung sections stained with
hematoxylin and eosin were captured in a blinded manner using Leica QWin image software (Leica Microsystems, Wetslar, Germany)(4 animals/group and 6 random highpowered fields/animal). We report the airspace area as a percentage of mesenchyme area using morphometric techniques (MetaMorph 7.7 software, Molecular Devices
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Corporation, Sunnyvale, CA). To determine alveolar type II cell counts, TTF-1+ cells were counted in five random lung sections per animal (4 animals/group) by a blinded observer. The average number of positive cells per animal were used to compute group
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averages. Statistical Analysis
Values were expressed as mean ± SD. Groups were compared using ANOVA
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for normally distributed data and Kruskal-Wallis ANOVA for non-gaussian data. If the ANOVA was significant, post-hoc tests were used to compare the following groups: 1)
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Each group against control, and 2) Beta or LPS against Beta+LPS. Dunn’s or Bonferroni’s correction was applied as appropriate to adjust for multiple-comparisons.
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All p-values <0.05 were considered significant.
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RESULTS There was no maternal or fetal mortality. One of eight ewes in the maternal betamethasone group was in preterm labor at the time of surgical delivery. Fetal
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weights, lung/body weight ratios, cord blood neutrophil counts, cord blood pH were similar across groups but blood neutrophil counts increased with either betamethasone or LPS (Table 1).
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Fractional Airspace Area Measurements (Anatomic lung maturation)
Betamethasone treated animals had a 47% increase in airspace area relative to
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the control animals (p=0.028), with larger airspaces signifying mesenchymal thinning (Figure 1 A-B). The LPS exposure did not increase airspace area. LPS in combination with betamethasone caused a more variable response, perhaps due to variable components of lung inflammation and steroid responses.
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Surfactant protein and lipid synthesis genes (Molecular lung maturation) Surfactant proteins A and D have innate host defense functions, while surfactant proteins B and C decrease surface tension.25 Beta exposure increased mRNAs for SP-
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A and SP-D but not SP-B and SP-C (Figure 2 A--D). The LPS exposure did not increase any surfactant protein mRNAs. The effects of the combined exposures were
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comparable to Beta alone. Treatment with Beta or LPS separately had no effect on the surfactant lipid processing enzymes CEPT1, CCTα, or LPCAT-1 expression (Figure 2E,F,H)(see figure legend for function of the enzymes). The mRNA for FASN was increased by Beta but not LPS, and the combination reflected beta effects (Figure 2G). Treatment with Beta and LPS in combination, significantly increased mRNA levels of LPCAT1 (Figure 2H).
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Alveolar type II cells (Cellular lung maturation) Thyroid transcription factor-1 (TTF-1) positive cells identify type II alveolar epithelial cells and pro-Surfactant protein C (pro-SPC) expression signify mature alveolar type II
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epithelial cells.26 There were no differences in the numbers of TTF-1+ cells across treatment groups (data not shown). Pro-SPC expression was not detected in any of the treatment groups indicating immaturity of the type II cells (data not shown).
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Expression of corticosteroid receptors
The mRNA expression of glucocorticoid receptors Nuclear Receptor Subfamily 3
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Group C Members 1 and 2 (NR3C1) and (NR3C2) significantly decreased following treatment with Beta (NR3C1, Control 1.0±0.3 vs Beta 0.6±0.2; NR3C2 Control 1.0±0.3 vs Beta 0.5±0.1, p<0.05, n=8/group). LPS had no effect on mRNA expression of either mRNA, with no additional effects of both exposures.
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Fetal lung liquid regulation
Epithelial sodium channel (SCNN1-A or eNaC-alpha, SCNN1-B or eNaC-beta and SCNN1-G or or eNaC-gamma subunits), Na-K ATPase (ATP1B1), and Aquaporin
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are required for postnatal lung fluid clearance.27, 28 The mRNA expression of SCNN1-B and SCNN1-G subunits was significantly increased following maternal treatment with
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Beta (Figure 3 A-C).
SCNN1A mRNA only increased with Beta+LPS exposure.
Treatment with Beta significantly decreased mRNA of ATP1B1, with no effect from LPS (Figure 3D). There was no significant increase in mRNA expression of Aquaporin 5 (AQP5) with Beta (Figure 3E). In general, Beta plus LPS exposure groups had similar changes in epithelial fluid clearance genes compared to Beta alone.
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mRNA expression of antioxidants, vascular mediators and acute phase response genes. We quantified gene expression of pathways known to be induced by either Beta
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or LPS in the more mature 125d gestation sheep fetus.29, 30 There were no changes in the mRNAs for the anti-oxidant enzymes Superoxide dismutase 2 (SOD2), Catalase (CAT) and Glutatione peroxidase 1 (GPX1) (Table 2). Similarly, the mRNAs for
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vascular growth factor VEGF-A isoform 165 and 185 and NOS3 were also not altered by these exposures. The mRNA for the acute phase response genes Connective tissue
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growth factor (CTGF), Cysteine rich angiogenic inducer 61 (CYR61), Early growth response protein-1 (EGR1), and Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A) were not changed by either Beta, LPS or the combined exposures (Table 2). Lung inflammation
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As anticipated, LPS significantly increased mRNA expression of the inflammatory cytokines, IL-1β, IL-8, IL-6, TNF-α, and MCP-1 (Figure 4 A-E). Treatment with Beta did not decrease LPS induced cytokine mRNA expression (non-significant in ANOVA with
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post hoc comparison due to adjustment with multiple group comparisons, but significant for a two group comparison). Myeloperoxidase expressed by activated neutrophils
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were
of
seen
in
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rarely
any
group
(data
not
shown).
The
presence
monocytes/macrophages in the fetal lung was assessed using PU.1, since it is a marker of activated monocytes and is expressed during maturation of macrophages
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.
Macrophages were not detected in the fetal lungs in any of the groups (data not shown). Chorioamnion and systemic inflammation
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LPS significantly increased mRNA expression of the inflammatory cytokines, IL1β, IL-8, TNF-α but not IL6 (Figure 5 A-E). Beta did not signficantly decrease LPS responses on cytokine/chemokine mRNAs. Surprisingly, Beta but not LPS increased
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mRNAs for Serum amyloid A3 (Figure 5 F-G).
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mRNAs for C-reactive protein (CRP) in the fetal liver, and both Beta and LPS increased
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DISCUSSION Principal Findings A comprehensive evaluation of fetal lung maturation to antenatal steroids in the
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peri-viable period demonstrated that the major effect of betamethasone in the extremely preterm sheep was mesenchymal thinning with a resultant increase in air-space, the magnitude of which was comparable to the 125d gestation sheep.18 However, unlike the
lung maturation did not increase.
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more mature sheep, surfactant protein B or surfactant protein C mRNA, indicators of Together, these results demonstrate a partial
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maturational response in the fetus equivalent to the peri-viable human fetus compared to those in the more mature preterm sheep lung.
Meaning of the results in the context of what is known - equivalence of sheep to human fetal gestation age
The 94d fetal gestation was selected for the study to be at the late cannalicular
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period of lung structural development that corresponds with about weeks 20-23 in the human fetus. The original studies by Liggins,34 and subsequent experimental work35
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that formed the basis of antenatal steroid treatment was done in approximately 125 d gestation sheep. The fetal sheep lung matures structurally to begin alveolarization at
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about 116 days or 0.77 of term while in the human alveolarization begins only in late fetal life - 0.89 of term gestation.36
In contrast, the fetal human lung can secrete
surfactant by 22 weeks (0.55 of term gestation) while the fetal sheep lung is quite surfactant deficient until after about 125 days' gestation (0.83 term gestation).36,
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Accepting these limitations of the timing of different components of lung maturation, our study provides new in vivo information about lung maturation and inflammation responses revelent to the peri-viable period in humans.
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Functional implications of the study 1) Anatomic maturation - The decrease in Betamethasone induced mesenchymal thinning would be expected to increase lung compliance 38. Improved fetal lung
thinning
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fluid transport and lung remodeling could both explain the mesenchymal . Although antenatal steroids inhibit alveolar septation,40 the short-
term effect is better aeration of the preterm lung.
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2) Molecular maturation of the surfactant system - The extremely preterm sheep did not increase mRNAs for the surfactant protein B and C with either Beta or
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LPS as in the 125d gestation sheep.41 However, Beta induced increases in mRNAs for the innate host defense proteins SP-A and SP-D were similar to the responses to the more mature sheep.41 Consistent with no induction of SP-C mRNA by Beta or LPS in the extremely immature sheep, TTF1 positive type II
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cells were not proSP-C positive. The Betamethasone exposure increased only 1 of the 4 measured mRNAs in lipid synthetic pathways with no response to LPS. Overall these findings indicate that surfactants are minimally induced by
fetus.
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antenatal steorids and not induced by LPS in the extremely immature sheep
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3) Fetal lung fluid clearance, antioxidants, and steroid receptors – The mRNAs for the SCNN1 B and G subunits of the epithelium sodium channel increased, while ATP1B1 (Na-K ATPase) decreased and Aquaporin 5 remained unchanged, indicating incomplete responses in epithelial fluid clearance genes. The antioxidant, vascular, and early response gene expression in the fetal lungs were unaltered by Beta.
Curiously, Beta down-regulated 2 steroid
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receptors. The general pattern indicates a partial response of the extremely preterm lung to Beta and essentially no response to LPS. 4) Lung inflammation - In these extremely preterm sheep, intraamniotic LPS
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induced large increases in pro-inflammatory cytokine responses in the fetal lung, but no inflammatory cell infiltrate was apparent. This result contrasts with large increases in airway neutrophils in the lung in the 125d gestation sheep.26
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Blood neutrophil counts, indicators of systemic inflammation increased in the extremely preterm sheep similar to effects in the 125d gestation sheep.42
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Intraamniotic LPS induced large increases in the inflammatory cytokine mRNAs in the chorioamnion but the systemic inflammatory responses in the liver were more modest. The unanticipated result was Beta induced mRNAs for both serum amyoid A3 and CRP, widely used markers of systemic
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inflammation. Although Beta is known to mature the function of monocytes,32 the induction of acute phase reactants by Beta has not been reported to our knowledge.
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5) Effects of combined exposure to Betamethasone and LPS - The combined exposure
of
the
anti-inflammatory
Beta
at
the
same
time
as
the
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proinflammatory LPS augments the maturational effects of either stimulus in older gestational age sheep.26,
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However, this additive effect of Beta and
intraamniotic LPS was not detected in the 94 day gestation sheep. In contrast to potent inflammation suppressive effects at 125d gestation, Beta incompletely blunted the fetal lung cytokine response to LPS in the more immature sheep.22
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Thus, the more immature lung is less responsive overall than the more mature lung, both in terms of lung inflammation and lung maturation. Clinical Implications
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Currently ACOG rates the recommendation to give antenatal steroids at 230 to 236 d gestation as “weak” (level 2B) and at 240 or greater as strong with moderate quality evidence (level 1B) 9. We demonstrate some benefits of antenatal steroids in the
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sheep lung that is anatomically similar to the peri-viable human gestation (larger airspace in the lung due to anatomic maturation) but no benefical effects on the surfactant
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system. While our results do not contradict the ACOG recommendations to give maternal steroids at the limits of viability, the lung benefits may be less than at older gestations. Research Implications
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We previously reported that Beta maximally increased fetal lung surfactant protein mRNAs at 1-2d with a return to baseline by 7d 21. In contrast, surfactant proteins and lipids increased in the airways between 2-7d after exposure
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. Time course of
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antenatal steroid effects may differ with extreme prematurity. Rabbit lung explant responses to steroids and inflammatory stimuli are gestation dependent
44, 45
. The dose
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for Beta and LPS were the same as those used previously by our group in the 125d gestation sheep and the Beta dose was higher than the clinical dose. Thus, the blunted response of the fetal lung at early gestation is unlikely to be explained by inadequate dose exposure. We recently reported that half the dose of Beta is also effective in inducing lung maturation in the 125d gestation sheep 46. Strengths and Weaknesses
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These results have some limitations for generalization.
The number of
observations per treatment group are limited with animal experiments. The exposures were for 48 hours to evaluate early maturational effects, and evaluations made at longer
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intervals after exposure may yield different results. In more mature sheep, Beta and LPS exposures given 7 days apart yielded similar lung maturation signals as evaluations at shorter intervals.47 Alternatively, the 48h exposure may have missed a
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critical window of response in the hours immediately following exposure. We recognize that occult chorioamnionitis associated with very preterm delivery will precede maternal therapy.
Thus,
the
experimental
design
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corticosteroid
of
giving
maternal
betamethasone and intraamniotic LPS at the same time is an unlikely clinical scenario and therefore a limitation of the study. The fetal exposures that result in very preterm deliveries are varied in cause and duration and conceptually all extremely preterm
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infants are not normal. In contrast, most animal models utilize normal pregnancies. A strength of our study is comprehensive evaluation of multiple different maturational pathways in the fetal lung.
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Conclusion
Within the limitations of our study, our results demonstrate partial maturational
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and inflammatory responses of the extremely preterm fetal sheep lung to maternal steroids and LPS. While the lung air-space fraction increased, surfactant protein-B and surfactant-protein C mRNAs did not increase, indicating minimal effects on the surfactant system.
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Acknowledgements: The authors wish to thank Manuel Alvarez Jr. for his excellent technical contributions, research personnel at the University of Australia sheep facility for their excellent help
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with animal husbandry.
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Figure Legends: Figure 1: Antenatal betamethasone (Beta) induced mesenchymal thinning increasing the relative air-space area in the fetal lung. Paraffin sections from inflation fixed fetal
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lungs were sectioned and stained with hematoxylin and eosin. Airspace area relative to mesenchyme area was measured using computerized morphometry in a blinded manner. (A) The average of 6 random high-power fields/animal was used as a
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representative value for the animal. (B) Representative H&E stained lung sections from
Bonferroni test, 4 animals/group).
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each group. (*p<0.05 vs control, #p<0.05 vs Beta+LPS by ANOVA with post-hoc
Figure 2: Antenatal betamethasone (Beta) partially induced genes essential for surfactant function in the fetal lung. Total RNA from fetal lung was used for quantitiative reverse-transcriptase and PCR amplification using Rhesus specific Taqman probes.
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The values were initially standardized to 18S RNA and the resultant values were expressed as fold-change relative to the average of control value assigned as 1. (A-D) Expression of Surfactant protein genes. (E-H) Expression of genes essential for
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surfactant lipid synthesis. Beta did not increase mRNAs for SFTPB (surfactant proteinB), SFTPC (surfactant protein C), CEPT1 (Choline/Ethanolamine phoshphotransferase
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1 which catalyzes the reaction of CDP-choline and diacylglycerol to form PC)
48
, CCTα
(Phosphocholine cytidyltransferase a which catalyzes the condensation of cytidine triphosphate (CTP)
and phosphocholine to form
CDP-choline)
49
or
LPCAT1
(Lysophosphatidylcholine acyltransferase which is a key enzyme in surfactant phosphatidyl choline remodeling to form dipalmitoylphosphatidylcholine, the major surface-active component of surfactant)
50
, but slightly increased FASN (Fatty acid
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synthase the enzyme for de novo synthesis of fatty acids used for Phosphatidylcholine synthesis)
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. Both Beta and LPS increased mRNAs for SFTPA (surfactant protein A)
and SFTPD (surfactant protein D) involved in innate host defense (*p<0.05 vs control,
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#p<0.05 vs Beta+LPS by Kruskall-Wallis ANOVA with post-hoc Dunn’s test, 8 animals/group).
Figure 3: Antenatal betamethasone (Beta) partially induced genes essential for
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epithelial fluid clearance in the fetal lung.
Total RNA from fetal lung was used for quantitiative reverse-transcriptase and PCR
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amplification using Rhesus specific Taqman probes. The values were initially standardized to 18S RNA and the resultant values were expressed as fold-change relative to the average of control value assigned as 1. Beta increased the mRNAs for SCNN1B, and SCNN1G but not SCNN1A, ATP1B1 or AQP5. LPS only had no effect.
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The combination exposure largely reflected Beta effects (*p<0.05 vs control, #p<0.05 vs Beta+LPS by Kruskall-Wallis ANOVA with post-hoc Dunn’s test, 8 animals/group). Figure 4: Intraamniotic LPS induced expression of inflammatory cytokine mRNAs in the
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fetal lung
Total RNA from fetal lung was used for quantitiative reverse-transcriptase and PCR
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amplification using Rhesus specific Taqman probes. The values were initially standardized to 18S RNA and the resultant values were expressed as fold-change relative to the average of control value assigned as 1. LPS increased the mRNAs for IL-1ß, IL-8, TNFα, and MCP-1. For IL-6, the ANOVA was significant but post-hoc tests did not show differences between groups. Beta tended (non-siginificant by ANOVA with post-hoc test, significant in a two group t-test comparison) to suppress LPS-induced
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inflammatory cytokine mRNAs (*p<0.05 vs control, #p<0.05 vs Beta+LPS by KruskallWallis ANOVA with post-hoc Dunn’s test, 8 animals/group). Figure 5: Intraamniotic LPS induced expression of inflammatory cytokine mRNAs in the
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chorioamnion and the fetal liver
Total RNA from chorioamnion (A-E) and fetal liver (F-G) was used for quantitiative reverse-transcriptase and PCR amplification using Rhesus specific Taqman probes.
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The values were initially standardized to 18S RNA and the resultant values were expressed as fold-change relative to the average of control value assigned as 1. LPS
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increased the mRNAs for IL-1ß, IL-8, TNFα, and MCP-1 in the chorioamnion. Beta tended (non-siginificant by ANOVA with post-hoc test, significant in a two group t-test comparison) to suppress LPS-induced inflammatory cytokine mRNAs. In the fetal liver, both LPS and Beta individually increased mRNAs for serum amyloid A3 in the fetal liver,
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and only Beta but not LPS increased CRP mRNA (*p<0.05 vs control, #p<0.05 vs
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Beta+LPS by Kruskall-Wallis ANOVA with post-hoc Dunn’s test, 8 animals/group).
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TABLE 1: Physiologic data of the animals at birth
Beta
Animal Number
8
8
Male/Female
4/4
3/5
Birth Weight (kg)
0.74±0.06
Cord Blood pH
LPS
Beta + LPS
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Control
8
5/3
5/3
0.73±0.07
0.73±0.03
0.65±0.08
7.22±0.07
7.18±0.07
7.27±0.03
7.26±0.09
Lung Weight/Birth Weight (g/kg)
44.8±4.4
43.7±7.6
43.4±9.7
43.5±3.4
Hematocrit (%)
31±2
30±3
30±5
30±3
1.2±0.5
1.6±0.5
1.3±0.3
1.3±0.3
0.09±0.03
0.5±0.4*
0.3±0.1*
0.4±0.2*
0.8±0.3
0.9±0.3
0.9±0.3
0.8±0.2
0.04±0.04
0.06±0.06
0.04±0.03
0.04±0.03
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Neutrophils (x109/l)
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Lymphocytes (x109/l) Monocytes (x109/l)
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Total WBC (x109/l)
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Values are mean ± SD. WBC, White Blood Cells. *p<0.05 vs. control.
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TABLE 2: Effect of antenatal betamethasone on antioxidant, vascular, and acute response genes in the fetal lung Beta
LPS
Superoxide dismutase 2 (SOD2)
1.0±0.4
0.7±0.2
1.5±0.6
1.1±0.5
Catalase (CAT)
1.0±0.7
0.7±0.2
0.8±0.3
0.9±0.2
Glutathione peroxidase 1 (GPX1)
1.0±0.5
0.7±0.2
1.3±0.3
1.1±0.5
Antioxidant genes
Vascular endothelial growth factor A 1.0±0.3 (VEGF-165) Vascular endothelial growth factor A 1.0±0.3
Acute Response genes Connective
tissue
growth
1.2±0.3
0.8±0.2
1.0±0.4
1.1±0.3
0.8±0.2
0.9±0.4
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(VEGF-185)
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Vascular/Angiogenesis genes
Beta + LPS
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Control
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mRNA
factor 1.0±0.4
0.7±0.4
0.8±0.5
0.8±0.3
Cysteine-rich angiogenic inducer 61 1.0±0.4
0.6±0.3
0.9±0.4
0.6±0.1
1.0±0.4
0.7±0.3
0.9±0.3
0.7±0.2
Nuclear receptor subfamily 4, group 1.0±0.3
0.7±0.2
0.7±0.2
0.7±0.1
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(CTGF)
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(CYR61)
Early growth response 1 (EGR1)
A, member 1 (NR4A1)
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S0002-9378(17)32698-4
DOI:
10.1016/j.ajog.2017.12.207
Reference:
YMOB 11995
To appear in:
American Journal of Obstetrics and Gynecology
Received Date: 23 June 2017 Revised Date:
27 November 2017
Accepted Date: 14 December 2017
Please cite this article as: Visconti K, Senthamaraikannan P, Kemp MW, Saito M, Kramer BW, Newnham JP, Jobe AH, Kallapur SG, Extremely preterm fetal sheep lung responses to antenatal steroids and inflammation, American Journal of Obstetrics and Gynecology (2018), doi: 10.1016/ j.ajog.2017.12.207. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Extremely preterm fetal sheep lung responses to antenatal steroids
Kevin Visconti MD
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and inflammation
Paranthaman Senthamaraikannan PhD
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Matthew W. Kemp PhD Masatoshi Saito MD PhD
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Boris W. Kramer MD PhD John P. Newnham MD Alan H. Jobe MD PhD
Suhas G. Kallapur MD Disclosure: The authors report no conflict of interest
Contact information:
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Suhas G. Kallapur MD Professor of Pediatrics
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Funding: Financial Markets Foundation for Children Grant to MWK, MS and JPN.
Neonatal Research Center of the UCLA Children’s Discovery and Innovation Institute, Division of Neonatology & Developmental Biology, Department of Pediatrics, David
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Geffen School of Medicine UCLA. Mattel Children's Hospital UCLA 10833 Le Conte Avenue, Room B2-375 MDCC Los Angeles, CA 90095 Tel: 310-206-8489
Fax: 310-267-0154 Email id: [email protected] Word count: 2980
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Condensation: Fetal sheep lungs comparable to peri-viable human lungs have decreased maturational or inflammatory responses compared to more mature preterm
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sheep lungs.
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Short-version of the title: Effect of the antenatal steroid in the periviable fetal lung
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Glossary of terms:
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ATP1B1 - Sodium potassium ATPase (Na-K ATPase) (Ion channel) ANS - Antenatal steroids Beta - Betamethasone CAT - Catalase (antioxidant enzyme) CCTα - Phosphocholine cytidyltransferase a (an intermediary in the surfactant lipid pathway) CDP-choline Cytidine diphosphate choline (an intermediary in the surfactant lipid pathway) CEPT1 - Choline/Ethanolamine phoshphotransferase 1 (an intermediary in the surfactant lipid pathway) CTGF - Connective tissue growth factor CRP - C-reactive protein (an acute phase reactant) CTP - Cytidine triphosphate CYR61 - Cysteine rich angiogenic inducer 61 (an acute phase reactant) DPPC - dipalmitoylphosphatidylcholine (component of surfactant lipid) EGR1- Early growth response protein-1 FASN - Fatty acid synthase (enzyme in the surfactant lipid synthesis pathway) GPX1 - Glutathione peroxidase (antioxidant enzyme) IL1beta - Interleukin 1 beta IL6 - Interleukin 6 IL8 - Interleukin 8 LPCAT1- Lysophosphatidylcholine acyltransferase (enzyme in the surfactant lipid synthesis pathway) LPS - lipopolysaccharide MCP1 - Monocyte chemoattractant protein 1 MPO - Myeloperoxidase (used as a neutrophil marker) Na-K ATPase - Sodium Potassium ATPase (ion channel) NOS - Nitric oxide synthase NR3C - Nuclear Receptor Subfamily 3 Group C (steroid receptor) NR4A - Nuclear Receptor Subfamily 4 Group A Member 1 PC - Phosphatidyl choline (intermediary in surfactant lipid) PU.1 - A transcription factor that is expressed in maturing monocytes/macrophages RT-PCR - Reverse transcriptase polymerase chain reaction Pro-SPC - A precursor protein of surfactant protein C SCNN1- Epithelial sodium channel (formerly called eNac) SOD - Superoxide dismutase (antioxidant enzyme) SP-A - Surfactant protein A SP-B - Surfactant protein B SP-C - Surfactant protein C SP-D - Surfactant protein D TNFa - Tumor necrosis factor alpha TTF-1 - Thyroid transcription factor 1 (a marker of alveolar type II cell) VEGF - Vascular endothelial growth factor
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ABSTRACT 244 words Background: The efficacy of antenatal steroids for fetal lung maturation in the peri-
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viable period is not fully understood.
Objective(s): To determine the lung maturational effects of ANS and inflammation in early gestation sheep fetuses, similar to the peri-viable period in humans.
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Study Design: Date-mated ewes with singleton fetuses were randomly assigned to one of 4 treatment groups (n=8/group): 1) maternal intramuscular injection (IM) of
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betamethasone (Beta), 2) intra-amniotic lipopolysaccharide (IA) (LPS), 3) Beta+LPS, 4) IA+IM Saline (controls). Fetuses were delivered surgically 48h later at 94d gestation (63% term gestation) for comprehensive evaluations of lung maturation, lung and systemic inflammation.
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Results: Relative to controls, 1) Betamethasone increased the fetal lung air-space tomesenchymal area ratio by 47% but did not increase the mRNAs for the surfactant
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proteins (SP) B and C that are important for surfactant function or increase the expression of pro-SP-C in the alveolar type II cells. 2) Betamethasone increased
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expression of 1 of the 4 genes in surfactant lipid synthetic pathways, 3) Betamethasone increased genes involved in epithelium sodium channel transport, but not Na-K ATPase or Aquaporin 5. 4) Lipopolysaccharide increased pro-inflammatory genes in the lung but did not effectively recruit activated inflammatory cells. 5) Betamethasone incompletely suppressed Lipopolysaccharide induced lung inflammation. In the liver, Betamethasone when given alone increased the expression of serum amyloid A3 and C-reactive protein mRNAs.
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Conclusion(s): Compared the more mature 125d gestation sheep, antenatal steroids do not induce pulmonary surfactants during the peri-viable period, indicating a different
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response.
Key words: fetal lung maturation, chorioamnionitis, surfactant, respiratory distress
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syndrome, fetal inflammation
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INTRODUCTION Antenatal steroids (ANS) for at risk pregnancies from 24 to 34 weeks gestational age are the standard of care since the NIH consensus statement of 1995.1 However,
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only about 100 women treated with ANS delivered before 28 weeks in all of the single randomized trials performed before 1993.2 Thus, there are limited high quality data to support benefit of ANS at early gestation despite increasing populations of these
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infants.3 Several large observational studies recently demonstrated efficacy of ANS at the margins of viability.4-8 The ACOG/SMFM Obstetrical Clinical Consensus (October
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2017) states that antenatal steroids are not recommended prior to 23w0d, may be "considered" at 23w0d, and are recommended for 24w0d gestation and beyond 9. However, there may be an inherent selection bias in outcomes in the observational studies since pregnancies in the peri-viable period given ANS likely receive more
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survival directed care.
Although early gestation human lung explants and fetal lung cells in culture respond to corticosteroids with morphologic changes and induction of surfactant
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components,10 whether these changes occur in vivo is not known. ANS increase lung volumes in fetal monkeys at the margin of viability, but the effects on surfactant system
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are not known.11 Thus, the question of response characterisitics and efficacy of ANS at the margins of viability is not fully settled. Fetal exposure to chorioamnioitis can decrease the incidence of respiratory
distress syndrome by inducing early lung maturation clinically,
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a phenomenon also
demonstrated in animal models.13 In fetal sheep models, the combined fetal exposure to inflammation and corticosteroids induce more lung maturation than either stimulus
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alone.14 However, either exposure may have adverse fetal effects: fetal inflammatory responses with fetal injury for inflammation and decreased fetal growth and concerns about neurodevelopment for corticosteroids 15, 16.
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Although there are some studies at earlier gestational ages, most of the randomized clinical data and experimental data in sheep for effects of antenatal steroids are for more than 80% term gestation.2,17, 18 Despite their common occurences, there is
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lack of information regarding early gestation effects of combined exposures to antenatal steroids and inflammation on the fetal lung 5, 19. We hypothesized that, in early gestation
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fetal sheep, both ANS and intra-uterine inflammation would increase indicators of lung
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maturation and the two exposures would further increase the maturational signals.
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METHODS Study Design With the approval of Animal Ethics Committee at The University of Western
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Australia, time-mated ewes with singleton fetuses were randomly assigned to one of 4 treatment groups: 1) Intra-amniotic injection of lipopolysaccharide (LPS) (10 mg Escherichia coli 055:B5, Sigma Chemical, St. Louis, MO), 2) Maternal intramuscular
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injection of betamethasone 0.5 mg/kg maternal weight [Celestone Soluspan, ScheringPlough, New South Wales, Australia], 3) Intra-amniotic LPS and betamethasone in
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combination, or 4) Intra-amniotic + intra-muscular injections of saline (controls) at 92 days GA (n=8 animals/group). This betamethasone dose and duration of exposure of 2d was based on previous studies demonstrating increases in surfactant protein-B and surfactant protein-C mRNA in Beta exposed 124d gestation sheep
20, 21
, but the
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experimental dose is higher than what is used clinically. The maternal betamethasone was given about 3 h prior to the intra-amniotic injection of LPS
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to allow time for fetal
exposure of Beta prior to IA LPS. This experimental design reported previously
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allowed comparison of effects in the 124d gestation to the 94d gestation sheep. Some animals were used for fetal a pulmonary vascular study 23.
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Animal sampling protocol
Lambs were surgically delivered 2d after exposures at 94d GA and euthanized
with 100 mg/kg pentobarbital. Each fetus was weighed and fetal cord blood was collected for cell counts. The lungs were removed, separated, and weighed. Lung tissue from the right lower lobe was snap frozen and the right upper lobe was inflation fixed in 10% buffered formalin at 30 cm H2O pressure for 24 h. The very immature late
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canalicular lung structure will not support ventilation for physiologic measurements of lung mechanics. Messenger RNA Quantification
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Total RNA extraction from frozen tissue samples and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed as previously described to quantify expression of key genes involved in surfactant function, lung fluid
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homeostasis, and lung inflammation. 24 Immunohistochemistry
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Immunohistochemistry assessed the distribution of alveolar type II cells and inflammatory cells in the fetal lung
24
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Briefly, the sections were incubated with
antibodies against Myeloperoxidase (Cell Marque, Rocklin, CA, cat # 289A-75, dilution 1:200), PU.1 (Santa Cruz Biotechnology, Santa Cruz, CA, cat # sc-352, dilution 1:500)
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TTF-1 (Seven Hills, Cincinnati, OH, cat # WMAb--8G7G31, dilution 1:500), Pro-SPC (Seven Hills, Cincinnati, OH, cat # R460, dilution 1:500) in 2% serum at 4oC overnight. Immunostaining was visualized using a Vectastain ABC peroxidase kit (Vector
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Laboratories Inc., Burlingame, CA). Slides were counterstained with Nuclear Fast Red for photomicroscopy.
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Lung Morphometry and alveolar type II cell counts To determine air-space fraction, images of lung sections stained with
hematoxylin and eosin were captured in a blinded manner using Leica QWin image software (Leica Microsystems, Wetslar, Germany)(4 animals/group and 6 random highpowered fields/animal). We report the airspace area as a percentage of mesenchyme area using morphometric techniques (MetaMorph 7.7 software, Molecular Devices
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Corporation, Sunnyvale, CA). To determine alveolar type II cell counts, TTF-1+ cells were counted in five random lung sections per animal (4 animals/group) by a blinded observer. The average number of positive cells per animal were used to compute group
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averages. Statistical Analysis
Values were expressed as mean ± SD. Groups were compared using ANOVA
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for normally distributed data and Kruskal-Wallis ANOVA for non-gaussian data. If the ANOVA was significant, post-hoc tests were used to compare the following groups: 1)
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Each group against control, and 2) Beta or LPS against Beta+LPS. Dunn’s or Bonferroni’s correction was applied as appropriate to adjust for multiple-comparisons.
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All p-values <0.05 were considered significant.
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RESULTS There was no maternal or fetal mortality. One of eight ewes in the maternal betamethasone group was in preterm labor at the time of surgical delivery. Fetal
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weights, lung/body weight ratios, cord blood neutrophil counts, cord blood pH were similar across groups but blood neutrophil counts increased with either betamethasone or LPS (Table 1).
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Fractional Airspace Area Measurements (Anatomic lung maturation)
Betamethasone treated animals had a 47% increase in airspace area relative to
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the control animals (p=0.028), with larger airspaces signifying mesenchymal thinning (Figure 1 A-B). The LPS exposure did not increase airspace area. LPS in combination with betamethasone caused a more variable response, perhaps due to variable components of lung inflammation and steroid responses.
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Surfactant protein and lipid synthesis genes (Molecular lung maturation) Surfactant proteins A and D have innate host defense functions, while surfactant proteins B and C decrease surface tension.25 Beta exposure increased mRNAs for SP-
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A and SP-D but not SP-B and SP-C (Figure 2 A--D). The LPS exposure did not increase any surfactant protein mRNAs. The effects of the combined exposures were
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comparable to Beta alone. Treatment with Beta or LPS separately had no effect on the surfactant lipid processing enzymes CEPT1, CCTα, or LPCAT-1 expression (Figure 2E,F,H)(see figure legend for function of the enzymes). The mRNA for FASN was increased by Beta but not LPS, and the combination reflected beta effects (Figure 2G). Treatment with Beta and LPS in combination, significantly increased mRNA levels of LPCAT1 (Figure 2H).
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Alveolar type II cells (Cellular lung maturation) Thyroid transcription factor-1 (TTF-1) positive cells identify type II alveolar epithelial cells and pro-Surfactant protein C (pro-SPC) expression signify mature alveolar type II
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epithelial cells.26 There were no differences in the numbers of TTF-1+ cells across treatment groups (data not shown). Pro-SPC expression was not detected in any of the treatment groups indicating immaturity of the type II cells (data not shown).
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Expression of corticosteroid receptors
The mRNA expression of glucocorticoid receptors Nuclear Receptor Subfamily 3
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Group C Members 1 and 2 (NR3C1) and (NR3C2) significantly decreased following treatment with Beta (NR3C1, Control 1.0±0.3 vs Beta 0.6±0.2; NR3C2 Control 1.0±0.3 vs Beta 0.5±0.1, p<0.05, n=8/group). LPS had no effect on mRNA expression of either mRNA, with no additional effects of both exposures.
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Fetal lung liquid regulation
Epithelial sodium channel (SCNN1-A or eNaC-alpha, SCNN1-B or eNaC-beta and SCNN1-G or or eNaC-gamma subunits), Na-K ATPase (ATP1B1), and Aquaporin
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are required for postnatal lung fluid clearance.27, 28 The mRNA expression of SCNN1-B and SCNN1-G subunits was significantly increased following maternal treatment with
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Beta (Figure 3 A-C).
SCNN1A mRNA only increased with Beta+LPS exposure.
Treatment with Beta significantly decreased mRNA of ATP1B1, with no effect from LPS (Figure 3D). There was no significant increase in mRNA expression of Aquaporin 5 (AQP5) with Beta (Figure 3E). In general, Beta plus LPS exposure groups had similar changes in epithelial fluid clearance genes compared to Beta alone.
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mRNA expression of antioxidants, vascular mediators and acute phase response genes. We quantified gene expression of pathways known to be induced by either Beta
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or LPS in the more mature 125d gestation sheep fetus.29, 30 There were no changes in the mRNAs for the anti-oxidant enzymes Superoxide dismutase 2 (SOD2), Catalase (CAT) and Glutatione peroxidase 1 (GPX1) (Table 2). Similarly, the mRNAs for
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vascular growth factor VEGF-A isoform 165 and 185 and NOS3 were also not altered by these exposures. The mRNA for the acute phase response genes Connective tissue
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growth factor (CTGF), Cysteine rich angiogenic inducer 61 (CYR61), Early growth response protein-1 (EGR1), and Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A) were not changed by either Beta, LPS or the combined exposures (Table 2). Lung inflammation
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As anticipated, LPS significantly increased mRNA expression of the inflammatory cytokines, IL-1β, IL-8, IL-6, TNF-α, and MCP-1 (Figure 4 A-E). Treatment with Beta did not decrease LPS induced cytokine mRNA expression (non-significant in ANOVA with
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post hoc comparison due to adjustment with multiple group comparisons, but significant for a two group comparison). Myeloperoxidase expressed by activated neutrophils
31
were
of
seen
in
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rarely
any
group
(data
not
shown).
The
presence
monocytes/macrophages in the fetal lung was assessed using PU.1, since it is a marker of activated monocytes and is expressed during maturation of macrophages
32
.
Macrophages were not detected in the fetal lungs in any of the groups (data not shown). Chorioamnion and systemic inflammation
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LPS significantly increased mRNA expression of the inflammatory cytokines, IL1β, IL-8, TNF-α but not IL6 (Figure 5 A-E). Beta did not signficantly decrease LPS responses on cytokine/chemokine mRNAs. Surprisingly, Beta but not LPS increased
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mRNAs for Serum amyloid A3 (Figure 5 F-G).
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mRNAs for C-reactive protein (CRP) in the fetal liver, and both Beta and LPS increased
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DISCUSSION Principal Findings A comprehensive evaluation of fetal lung maturation to antenatal steroids in the
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peri-viable period demonstrated that the major effect of betamethasone in the extremely preterm sheep was mesenchymal thinning with a resultant increase in air-space, the magnitude of which was comparable to the 125d gestation sheep.18 However, unlike the
lung maturation did not increase.
33
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more mature sheep, surfactant protein B or surfactant protein C mRNA, indicators of Together, these results demonstrate a partial
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maturational response in the fetus equivalent to the peri-viable human fetus compared to those in the more mature preterm sheep lung.
Meaning of the results in the context of what is known - equivalence of sheep to human fetal gestation age
The 94d fetal gestation was selected for the study to be at the late cannalicular
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period of lung structural development that corresponds with about weeks 20-23 in the human fetus. The original studies by Liggins,34 and subsequent experimental work35
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that formed the basis of antenatal steroid treatment was done in approximately 125 d gestation sheep. The fetal sheep lung matures structurally to begin alveolarization at
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about 116 days or 0.77 of term while in the human alveolarization begins only in late fetal life - 0.89 of term gestation.36
In contrast, the fetal human lung can secrete
surfactant by 22 weeks (0.55 of term gestation) while the fetal sheep lung is quite surfactant deficient until after about 125 days' gestation (0.83 term gestation).36,
37
Accepting these limitations of the timing of different components of lung maturation, our study provides new in vivo information about lung maturation and inflammation responses revelent to the peri-viable period in humans.
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Functional implications of the study 1) Anatomic maturation - The decrease in Betamethasone induced mesenchymal thinning would be expected to increase lung compliance 38. Improved fetal lung
thinning
39
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fluid transport and lung remodeling could both explain the mesenchymal . Although antenatal steroids inhibit alveolar septation,40 the short-
term effect is better aeration of the preterm lung.
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2) Molecular maturation of the surfactant system - The extremely preterm sheep did not increase mRNAs for the surfactant protein B and C with either Beta or
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LPS as in the 125d gestation sheep.41 However, Beta induced increases in mRNAs for the innate host defense proteins SP-A and SP-D were similar to the responses to the more mature sheep.41 Consistent with no induction of SP-C mRNA by Beta or LPS in the extremely immature sheep, TTF1 positive type II
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cells were not proSP-C positive. The Betamethasone exposure increased only 1 of the 4 measured mRNAs in lipid synthetic pathways with no response to LPS. Overall these findings indicate that surfactants are minimally induced by
fetus.
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antenatal steorids and not induced by LPS in the extremely immature sheep
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3) Fetal lung fluid clearance, antioxidants, and steroid receptors – The mRNAs for the SCNN1 B and G subunits of the epithelium sodium channel increased, while ATP1B1 (Na-K ATPase) decreased and Aquaporin 5 remained unchanged, indicating incomplete responses in epithelial fluid clearance genes. The antioxidant, vascular, and early response gene expression in the fetal lungs were unaltered by Beta.
Curiously, Beta down-regulated 2 steroid
16
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receptors. The general pattern indicates a partial response of the extremely preterm lung to Beta and essentially no response to LPS. 4) Lung inflammation - In these extremely preterm sheep, intraamniotic LPS
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induced large increases in pro-inflammatory cytokine responses in the fetal lung, but no inflammatory cell infiltrate was apparent. This result contrasts with large increases in airway neutrophils in the lung in the 125d gestation sheep.26
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Blood neutrophil counts, indicators of systemic inflammation increased in the extremely preterm sheep similar to effects in the 125d gestation sheep.42
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Intraamniotic LPS induced large increases in the inflammatory cytokine mRNAs in the chorioamnion but the systemic inflammatory responses in the liver were more modest. The unanticipated result was Beta induced mRNAs for both serum amyoid A3 and CRP, widely used markers of systemic
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inflammation. Although Beta is known to mature the function of monocytes,32 the induction of acute phase reactants by Beta has not been reported to our knowledge.
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5) Effects of combined exposure to Betamethasone and LPS - The combined exposure
of
the
anti-inflammatory
Beta
at
the
same
time
as
the
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proinflammatory LPS augments the maturational effects of either stimulus in older gestational age sheep.26,
43
However, this additive effect of Beta and
intraamniotic LPS was not detected in the 94 day gestation sheep. In contrast to potent inflammation suppressive effects at 125d gestation, Beta incompletely blunted the fetal lung cytokine response to LPS in the more immature sheep.22
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Thus, the more immature lung is less responsive overall than the more mature lung, both in terms of lung inflammation and lung maturation. Clinical Implications
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Currently ACOG rates the recommendation to give antenatal steroids at 230 to 236 d gestation as “weak” (level 2B) and at 240 or greater as strong with moderate quality evidence (level 1B) 9. We demonstrate some benefits of antenatal steroids in the
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sheep lung that is anatomically similar to the peri-viable human gestation (larger airspace in the lung due to anatomic maturation) but no benefical effects on the surfactant
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system. While our results do not contradict the ACOG recommendations to give maternal steroids at the limits of viability, the lung benefits may be less than at older gestations. Research Implications
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We previously reported that Beta maximally increased fetal lung surfactant protein mRNAs at 1-2d with a return to baseline by 7d 21. In contrast, surfactant proteins and lipids increased in the airways between 2-7d after exposure
38
. Time course of
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antenatal steroid effects may differ with extreme prematurity. Rabbit lung explant responses to steroids and inflammatory stimuli are gestation dependent
44, 45
. The dose
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for Beta and LPS were the same as those used previously by our group in the 125d gestation sheep and the Beta dose was higher than the clinical dose. Thus, the blunted response of the fetal lung at early gestation is unlikely to be explained by inadequate dose exposure. We recently reported that half the dose of Beta is also effective in inducing lung maturation in the 125d gestation sheep 46. Strengths and Weaknesses
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These results have some limitations for generalization.
The number of
observations per treatment group are limited with animal experiments. The exposures were for 48 hours to evaluate early maturational effects, and evaluations made at longer
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intervals after exposure may yield different results. In more mature sheep, Beta and LPS exposures given 7 days apart yielded similar lung maturation signals as evaluations at shorter intervals.47 Alternatively, the 48h exposure may have missed a
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critical window of response in the hours immediately following exposure. We recognize that occult chorioamnionitis associated with very preterm delivery will precede maternal therapy.
Thus,
the
experimental
design
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corticosteroid
of
giving
maternal
betamethasone and intraamniotic LPS at the same time is an unlikely clinical scenario and therefore a limitation of the study. The fetal exposures that result in very preterm deliveries are varied in cause and duration and conceptually all extremely preterm
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infants are not normal. In contrast, most animal models utilize normal pregnancies. A strength of our study is comprehensive evaluation of multiple different maturational pathways in the fetal lung.
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Conclusion
Within the limitations of our study, our results demonstrate partial maturational
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and inflammatory responses of the extremely preterm fetal sheep lung to maternal steroids and LPS. While the lung air-space fraction increased, surfactant protein-B and surfactant-protein C mRNAs did not increase, indicating minimal effects on the surfactant system.
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Acknowledgements: The authors wish to thank Manuel Alvarez Jr. for his excellent technical contributions, research personnel at the University of Australia sheep facility for their excellent help
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with animal husbandry.
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Figure Legends: Figure 1: Antenatal betamethasone (Beta) induced mesenchymal thinning increasing the relative air-space area in the fetal lung. Paraffin sections from inflation fixed fetal
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lungs were sectioned and stained with hematoxylin and eosin. Airspace area relative to mesenchyme area was measured using computerized morphometry in a blinded manner. (A) The average of 6 random high-power fields/animal was used as a
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representative value for the animal. (B) Representative H&E stained lung sections from
Bonferroni test, 4 animals/group).
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each group. (*p<0.05 vs control, #p<0.05 vs Beta+LPS by ANOVA with post-hoc
Figure 2: Antenatal betamethasone (Beta) partially induced genes essential for surfactant function in the fetal lung. Total RNA from fetal lung was used for quantitiative reverse-transcriptase and PCR amplification using Rhesus specific Taqman probes.
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The values were initially standardized to 18S RNA and the resultant values were expressed as fold-change relative to the average of control value assigned as 1. (A-D) Expression of Surfactant protein genes. (E-H) Expression of genes essential for
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surfactant lipid synthesis. Beta did not increase mRNAs for SFTPB (surfactant proteinB), SFTPC (surfactant protein C), CEPT1 (Choline/Ethanolamine phoshphotransferase
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1 which catalyzes the reaction of CDP-choline and diacylglycerol to form PC)
48
, CCTα
(Phosphocholine cytidyltransferase a which catalyzes the condensation of cytidine triphosphate (CTP)
and phosphocholine to form
CDP-choline)
49
or
LPCAT1
(Lysophosphatidylcholine acyltransferase which is a key enzyme in surfactant phosphatidyl choline remodeling to form dipalmitoylphosphatidylcholine, the major surface-active component of surfactant)
50
, but slightly increased FASN (Fatty acid
21
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synthase the enzyme for de novo synthesis of fatty acids used for Phosphatidylcholine synthesis)
49
. Both Beta and LPS increased mRNAs for SFTPA (surfactant protein A)
and SFTPD (surfactant protein D) involved in innate host defense (*p<0.05 vs control,
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#p<0.05 vs Beta+LPS by Kruskall-Wallis ANOVA with post-hoc Dunn’s test, 8 animals/group).
Figure 3: Antenatal betamethasone (Beta) partially induced genes essential for
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epithelial fluid clearance in the fetal lung.
Total RNA from fetal lung was used for quantitiative reverse-transcriptase and PCR
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amplification using Rhesus specific Taqman probes. The values were initially standardized to 18S RNA and the resultant values were expressed as fold-change relative to the average of control value assigned as 1. Beta increased the mRNAs for SCNN1B, and SCNN1G but not SCNN1A, ATP1B1 or AQP5. LPS only had no effect.
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The combination exposure largely reflected Beta effects (*p<0.05 vs control, #p<0.05 vs Beta+LPS by Kruskall-Wallis ANOVA with post-hoc Dunn’s test, 8 animals/group). Figure 4: Intraamniotic LPS induced expression of inflammatory cytokine mRNAs in the
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fetal lung
Total RNA from fetal lung was used for quantitiative reverse-transcriptase and PCR
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amplification using Rhesus specific Taqman probes. The values were initially standardized to 18S RNA and the resultant values were expressed as fold-change relative to the average of control value assigned as 1. LPS increased the mRNAs for IL-1ß, IL-8, TNFα, and MCP-1. For IL-6, the ANOVA was significant but post-hoc tests did not show differences between groups. Beta tended (non-siginificant by ANOVA with post-hoc test, significant in a two group t-test comparison) to suppress LPS-induced
22
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inflammatory cytokine mRNAs (*p<0.05 vs control, #p<0.05 vs Beta+LPS by KruskallWallis ANOVA with post-hoc Dunn’s test, 8 animals/group). Figure 5: Intraamniotic LPS induced expression of inflammatory cytokine mRNAs in the
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chorioamnion and the fetal liver
Total RNA from chorioamnion (A-E) and fetal liver (F-G) was used for quantitiative reverse-transcriptase and PCR amplification using Rhesus specific Taqman probes.
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The values were initially standardized to 18S RNA and the resultant values were expressed as fold-change relative to the average of control value assigned as 1. LPS
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increased the mRNAs for IL-1ß, IL-8, TNFα, and MCP-1 in the chorioamnion. Beta tended (non-siginificant by ANOVA with post-hoc test, significant in a two group t-test comparison) to suppress LPS-induced inflammatory cytokine mRNAs. In the fetal liver, both LPS and Beta individually increased mRNAs for serum amyloid A3 in the fetal liver,
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and only Beta but not LPS increased CRP mRNA (*p<0.05 vs control, #p<0.05 vs
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Beta+LPS by Kruskall-Wallis ANOVA with post-hoc Dunn’s test, 8 animals/group).
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TABLE 1: Physiologic data of the animals at birth
Beta
Animal Number
8
8
Male/Female
4/4
3/5
Birth Weight (kg)
0.74±0.06
Cord Blood pH
LPS
Beta + LPS
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Control
8
5/3
5/3
0.73±0.07
0.73±0.03
0.65±0.08
7.22±0.07
7.18±0.07
7.27±0.03
7.26±0.09
Lung Weight/Birth Weight (g/kg)
44.8±4.4
43.7±7.6
43.4±9.7
43.5±3.4
Hematocrit (%)
31±2
30±3
30±5
30±3
1.2±0.5
1.6±0.5
1.3±0.3
1.3±0.3
0.09±0.03
0.5±0.4*
0.3±0.1*
0.4±0.2*
0.8±0.3
0.9±0.3
0.9±0.3
0.8±0.2
0.04±0.04
0.06±0.06
0.04±0.03
0.04±0.03
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Neutrophils (x109/l)
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Lymphocytes (x109/l) Monocytes (x109/l)
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Total WBC (x109/l)
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8
Values are mean ± SD. WBC, White Blood Cells. *p<0.05 vs. control.
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TABLE 2: Effect of antenatal betamethasone on antioxidant, vascular, and acute response genes in the fetal lung Beta
LPS
Superoxide dismutase 2 (SOD2)
1.0±0.4
0.7±0.2
1.5±0.6
1.1±0.5
Catalase (CAT)
1.0±0.7
0.7±0.2
0.8±0.3
0.9±0.2
Glutathione peroxidase 1 (GPX1)
1.0±0.5
0.7±0.2
1.3±0.3
1.1±0.5
Antioxidant genes
Vascular endothelial growth factor A 1.0±0.3 (VEGF-165) Vascular endothelial growth factor A 1.0±0.3
Acute Response genes Connective
tissue
growth
1.2±0.3
0.8±0.2
1.0±0.4
1.1±0.3
0.8±0.2
0.9±0.4
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(VEGF-185)
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Vascular/Angiogenesis genes
Beta + LPS
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Control
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mRNA
factor 1.0±0.4
0.7±0.4
0.8±0.5
0.8±0.3
Cysteine-rich angiogenic inducer 61 1.0±0.4
0.6±0.3
0.9±0.4
0.6±0.1
1.0±0.4
0.7±0.3
0.9±0.3
0.7±0.2
Nuclear receptor subfamily 4, group 1.0±0.3
0.7±0.2
0.7±0.2
0.7±0.1
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(CTGF)
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(CYR61)
Early growth response 1 (EGR1)
A, member 1 (NR4A1)
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