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Intrapulmonary Instillation of Perflurooctylbromide Improves Lung Growth, Alveolarization, and Lung Mechanics in a Fetal Rabbit Model of Diaphragmatic Hernia Susanne Herber-Jonat, MD1; Aline Vuckovic, MD2; Rashmi Mittal, MD1; Anne Hilgendorff, MD1,3; Jacques C. Jani, MD4; Andreas W. Flemmer, MD1

Objectives: Fetal tracheal occlusion of hypoplastic rabbit lungs results in lung growth and alveolarization although the surfactant protein messenger RNA expression is decreased and the transforming growth factor-β pathway induced. The prenatal filling of healthy rabbit lungs with perfluorooctylbromide augments lung growth without suppression of surfactant protein synthesis. We hypothesizes that Intratracheal perfluorooctylbromide instillation improves lung growth, mechanics, and extracellular matrix synthesis in a fetal rabbit model of lung hypoplasia induced by diaphragmatic hernia. *See also p. 914. 1 Division of Neonatology, Dr. von Hauner Children’s Hospital, Perinatal Center Grosshadern, Ludwig-Maximilians-University Munich, Munich, Germany. 2 Laboratory of Physiology and Physiopathology, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium. 3 Comprehensive Pneumology Center, University Hospital, Ludwig-Maximilian-University and Helmholtz Centre, Munich, Germany. 4 Department of Obstetrics and Gynecology, University Hospital Brugmann, Brussels, Belgium. Dr. Flemmer is currently receiving a grant (FL-256/5-1) from the German Research Council (Deutsche Forschungsgemeinschaft). Dr. Flemmer provided expert testimony for the Bavarian Doctors Association and Stephan Inc.; lectured (teaching at seminars on mechanical ventilation and treatment for postgraduate doctors, e.g. Ipokrates Found); is employed by the University of Munich, Germany; and received support for travel (visiting academic at the Royal Women's Hospital Melbourne). His institution received grant support from the German Research Foundation and Medos Inc, Vygon Inc. Dr. HerberJonat lectured (seminars on mechanical ventilation and treatment for postgraduate doctors, e.g. Ipokrates Found) and is employed by the University of Munich. Her institution received grant support from the German Research Foundation. Dr. Vuckovic received grant support from Fonds de la Recherche Scientifique - FNRS (support for PhD student). Dr. Mittal’s institution received grant support from the German Research Foundation. Dr. Hilgendorff is employed by Ludwig-Maximilians University Munich and lectured for Milupa GmbH. Her institution received grant support, support for travel, and support for participation in review activities from Helmholtz Zentrum Muenchen. Dr. Jani disclosed that he does not have any potential conflicts of interest. Address requests for reprints to: Andreas W. Flemmer, MD, Division of Neonatology, Dr. von Hauner Children’s Hospital, Perinatal Center Grosshadern, Ludwig-Maximilian-University Munich, Munich, Germany. E-mail: [email protected] Copyright © 2014 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies DOI: 10.1097/PCC.0000000000000271

Pediatric Critical Care Medicine

Setting and Interventions: On day 23 of gestation, DH was induced by fetal surgery in healthy rabbit fetuses. Five days later, 0.8ml of perfluorooctylbromide (diaphragmatic hernia-perfluorooctylbromide) or saline (diaphragmatic hernia-saline) was randomly administered into the lungs of previously operated fetuses. After term delivery (day 31), lung mechanics, lung to body weight ratio, messenger RNA levels of target genes, assessment of lung histology, and morphological distribution of elastin and collagen were determined. Nonoperated fetuses served as controls. Measurements and Main Results: Fetal instillation of perfluorooctylbromide in hypoplastic lungs resulted in an improvement of lungto-body weight ratio (0.016 vs 0.013 g/g; p = 0.05), total lung capacity (23.4 vs 15.4 μL/g; p = 0.03), and compliance (2.4 vs 1.2 mL/cm H2O; p = 0.007) as compared to diaphragmatic herniasaline. In accordance with the results from lung function analysis, elastin staining of pulmonary tissue revealed a physiological distribution of elastic fiber to the tips of the secondary crests in the diaphragmatic hernia-perfluorooctylbromide group. Likewise, messenger RNA expression was induced in genes associated with extracellular matrix remodeling (matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2). Surfactant protein expression was similar in the diaphragmatic hernia-perfluorooctylbromide and diaphragmatic hernia-saline groups. Distal airway size, mean linear intercept, as well as airspace and tissue fractions were similar in diaphragmatic hernia-perfluorooctylbromide, diaphragmatic hernia-saline, and control groups. Conclusions: Fetal perfluorooctylbromide treatment improves lung growth, lung mechanics, and extracellular matrix remodeling in hypoplastic lungs, most probably due to transient pulmonary stretch, preserved fetal breathing movements, and its physical characteristics. Perfluorooctylbromide instillation is a promising approach for prenatal therapy of lung hypoplasia. (Pediatr Crit Care Med 2014; 15:e379–e388) Key Words: diaphragmatic hernia; lung mechanics; perfluorocarbon; pulmonary hypoplasia; surfactant protein; tracheal occlusion

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F

etal lung growth and maturation depend on fluid secretion and fetal breathing movements (1). During lung development, fluid is secreted continuously throughout gestation into the putative airways and is expelled into the surrounding amniotic fluid by fetal breathing movements (2). When the glottis is closed, the secretion of fluid generates a distending pressure sufficient to expand the developing lung to a certain extent (1). This periodic sequence of distending pressure, cyclical stretch, and equalization with the surrounding amniotic fluid appears to be one of the preconditions for normal lung development (3). A complete obstruction of the fetal airways results in massive lung distention (2). This observation led to the introduction of fetal tracheal balloon occlusion (TO) as a treatment option for lung hypoplasia due to congenital diaphragmatic hernia (DH). Here, the temporary closure of the trachea appears to significantly increase lung volume (4, 5) although no improvement of lung mechanics could be observed (6). In addition, prolonged TO results in a depletion of surfactant-producing type-II cells (5) and causes an increase in TGF-β2 expression (7). Thus, recent studies have advocated a plug-unplug procedure for TO in utero in order to prevent type-II cell depletion (8). However, this approach would subject neonates with severe DH to two procedures in utero. Animal experiments and human fetal MRI studies have reported that the greatest increase in lung volume and secondary crest formation occurs within 1–2 days of TO. Further increase in lung volume is mainly due to fluid accumulation, which disappears with removal of the tracheal occlusion (9). However, removal of a balloon soon after TO is considered high risk in human fetuses as it might result in preterm delivery with its concomitant complications. A reasonable alternative to balloon TO would be a one-step procedure that distends the lung temporarily but does not require a second procedure for removal. Ideally, the procedure should not prevent cyclic pressure and fluid equilibration between airways and amniotic cavity during fetal respiration. Perfluorocarbons have been believed to be inert, biocompatible substances with a specific weight that is 1.7 times higher compared to regular fetal pulmonary fluid (10). They are well known for their use as synthetic oxygen carriers in partial liquid ventilation, blood substitutes, and imaging agents due to their physical properties (10–12). Perfluoroctylbromide (PFOB), one of the intensively tested perfluorocarbons for biomedical use, has high solubility for oxygen (52.7 vol %), low vapor pressure (5 torr), and low surface tension (18 dynes/cm). PFOB is practically immiscible in water and has solubility in olive oil of 37 mM at room temperature (10, 12). Apart from their use as a respiratory medium, Nobuhara et al (13) demonstrated that perfluorocarbon distension of healthy lungs of neonatal lambs accelerated postnatal lung growth as evidenced by an increased alveolar number. In addition, postnatal filling of hypoplastic lungs with perfluorocarbon and consecutive partial liquid ventilation improved gas exchange and lung compliance in newborn lambs with DH (14). A similar effect with regard to gas exchange and accelerated lung growth was induced by postnatal instillation of perfluorocarbon into the lungs of human newborns with DH on extracorporeal membrane oxygenation (15–17). e380

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The feasibility of prenatal instillation of PFOB into the lungs of fetal rabbits has previously been studied (18–20). When instilled into healthy fetal rabbit lungs, PFOB increased lung growth without a reduction of surfactant protein B-positive cells as compared to TO (18, 20). We therefore hypothesized that prenatal instillation of PFOB into the hypoplastic lungs of rabbit fetuses with DH may improve lung growth and pulmonary mechanics in comparison to saline-treated animals without a concomitant decrease in surfactant protein gene levels.

MATERIALS AND METHODS Animal Subjects Experiments were performed in pregnant New Zealand white rabbits and their fetuses (21). All experiments were carried out in agreement with the guidelines of laboratory animals’ care and use of the U.S. National Institutes of Health and approval of the local animal care committee. Experimental Setup At day 23 of gestation, each rabbit doe and two of its fetuses underwent a surgical procedure under general anesthesia as previously described (21, 22). Briefly, the fetuses were subjected to an incision of the membranous part of the diaphragm through a left thoracotomy during an ex utero intrapartum (EXIT) procedure (21–23). Five days later, the previously operated fetuses were randomly subjected to intratracheal instillation of 0.8 mL sterile-filtered PFOB (DH-PFOB; Pharmpur, Augsburg, Germany) or saline (DH-saline) in a second EXIT procedure. For the instillation, the trachea was punctured with a 29-gauge cannula. After instillation of the respective fluid, the puncture was sealed with fibrin glue (TissueCol; Baxter, Eigenbrakel, Belgium) (24). Correct instillation of PFOB was confirmed by radioscopy immediately after fluid instillation (Fig. 1). Histological examinations of tracheas after the application of fibrin glue to the puncture did not show any signs of luminal obstruction or histological changes (data not shown). At term (day 31 of gestation), operated fetuses and nonoperated controls were delivered by cesarean section. Pilot experiments were carried out in order to define an adequate volume of PFOB to fill both lungs completely. The slow injection of 0.8 mL of PFOB resulted in a minimal reflux into the pharynx and an optimal filling of both lungs on radiographs on day 28. Study Population Statistical calculation estimated a sample size of 10 animals/ group in order to reach a significance level of 5% and statistical power of 80%. Referring to the statistical calculation and a suspected mortality rate of 50–60%, our randomized controlled study included 52 fetuses from 21 rabbit does. Forty-two fetuses were subjected to an incision of the diaphragm. Ten fetuses were assigned to serve as nonoperated controls. Five days after DH induction, 23 fetuses were still alive and received PFOB or saline instillation as randomized. Until delivery on day 31, another fetus died. There was no significant difference in mortality between the November 2014 • Volume 15 • Number 9

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into components representing the mechanical properties of the parenchyma and small airways (Rn, HL, and GL). All measurements were performed after calibration of the FlexiVent to account for pressure and flow drops due to tubing and endotracheal tube. Measurements with a coefficient of determination greater than or equal to 0.95 were accepted for analysis. Fetal Tissue Collection and Histology After lung function testing, the Figure 1. Representative fluoroscopic images of perflourooctylbromide (PFOB)-filled lungs of fetuses at day newborn rabbits were eutha28 and day 31 of gestation. The first examination was done after tracheal injection of 0.8 mL of PFOB on day nized; lungs were excised and 28 and replacement of the fetus in the uterus. The lungs as well as the trachea and the larynx are contrasted strongly by PFOB. Four days later, the lungs are still visible due to the faint contrast of PFOB. The arrow weighed. The lung-to-body indicates a small amount of PFOB in the stomach. weight ratio (LBWR) was calculated for the whole lung as well as each lung lobe separately. Whole lungs of three fetuses two groups, resulting in a sample size of 10 fetuses in the DHper group were expanded with formalin-free zinc fixative (BD PFOB group, 12 fetuses in the DH-saline group, and 10 nonopBiosciences, Heidelberg, Germany) and fixed under standarderated control fetuses, respectively. Two fetuses in the DH-saline group had to be excluded from the final analysis due to the devel- ized pressure (20 cm H2O) for 4 hours, after which the trachea was ligated and lungs were immersed in fixative for another opment of pneumothorax during mechanical ventilation. 20 hours before dehydration and paraffin embedding. Alveolar area, tissue and airspace fraction, as well as mean chord length Fetal Ventilation and Lung Mechanics were quantified using the Bioquant image analysis system Anesthetized fetuses were intubated for lung function analyafter isotropic uniform random sectioning (R&M Biometrics, ses under ventilatory support (volume-controlled ventilation Nashville, TN) (30). Relative amounts of insoluble lung elaswith 8 mL/kg body weight at a rate of 120 breath/min, 21% oxygen, end-expiratory pressure 3 cm H2O, room temperature, tin and collagen were assessed by quantitative image analysis and FlexiVent; SCIREQ, Montreal, QC, Canada) (22). Lung in Hart’s and trichrome-stained tissue slides, respectively (30). For consecutive messenger RNA (mRNA) analysis, lungs were mechanics were measured and calculated periodically (every excised and harvested in liquid nitrogen. 5 min) for 30 minutes using the custom-designed software (flexiWare; SCIREQ) of the FlexiVent ventilator. In order to RNA Extraction and Real-Time Quantitative measure respiratory mechanics, the ventilator briefly paused mechanical ventilation and executed different automated mea- Polymerase Chain Reaction RNA extraction was performed with RNAzol following the manusurement maneuvers, that is, “perturbations.” For the meafacturer’s protocol (WAK-Chemie Medical, Steinbach, Germany). surement of total lung capacity (TLC), the lungs were slowly RNA was purified using the RNAeasy protect minikit (Qiagen, inflated (3s) to a maximum inspiratory pressure of 30 cm Hilden, Germany), followed by complementary DNA (cDNA) H2O. During the following hold at peak pressure (3s), the lung volume reached an end-inspiratory plateau that reflected the synthesis using the First strand cDNA synthesis kit (GE HealthTLC (22). Automated pressure-volume (PV) loops were used care, Mickleton, NJ). Quantitative polymerase chain reaction to estimate the quasi-static mechanical properties of the respi- (PCR) was performed with 12.5 ng cDNA in a real-time PCR sysratory system. PV loops were generated by stepwise inflations tem (Step-one, Applied Biosystems, Life Technologies, Darmstadt, and deflations over 16s. Quasi-static elastance and compliance Germany). Primer pairs for target and reference genes were generated and validated as previously described (21). The resulting (Cst) as well as resistance values were automatically calculated by fitting the Salazar-Knowles equation to the PV loops (25, data were analyzed with the PCR Miner web tool (http://ewindup. info/miner/; Department of Biological Sciences and Program in 26). Newtonian resistance (Rn), tissue damping (GL), and tissue elastance (HL) were obtained using the forced oscillation Neuroscience, Stanford, CA) (31) to calculate the average PCR efficiency for each gene, and the results were processed using the technique as described elsewhere (22, 27, 28). Briefly, respiraREST software (http://www.REST.de.com; Qiagen) (32). The reftory input impedance (Zrs) in the frequency domain between 0.5 and 20 Hz by applying a 16-s composite signal contain- erence genes used were 36B4, topoisomerase 1 (TOP1), and succiing 19 mutually prime sinusoidal waves. The constant-phase nate dehydrogenase complex, subunit A (SDHA) as determined by model described by Hantos et al (29) was used to partition Zrs the geNORM software (Biogazelle, Gent, Belgium) (33). Pediatric Critical Care Medicine

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Statistical Analysis After testing for normal distribution, one-way analysis of variance with Student-Neuman-Keuls post hoc testing was performed. p values less than 0.05 were considered statistically significant. Results are presented as mean and sd, 95% CI, or sem as indicated (SPSS for Windows 21; IBM, Armonk, NY).

RESULTS Fetal Body Weight and LBWR Left-sided DH creation resulted in lung hypoplasia mainly on the left side indicated by the ratio of right/left LBWR in operated fetuses (DH-PFOB, 1.9 ± 0.5 and 1.9 ± 0.3 in DH-saline) as compared to controls (1.5 ± 1.2; p = 0.031). Likewise, the LBWR in DH-saline group was significantly lower compared with controls (0.013 ± 0.003 vs 0.023 ± 0.006; p < 0.001). Instillation of PFOB resulted in an increase of LBWR compared with DH-saline (0.016 ± 0.003; p = 0.05). Mean fetal body weight of DH-PFOB and DH-saline fetuses was not significantly different from that of controls (34.4 ± 6.6 g and 33.3 ± 6.7 g vs 32.9 ± 10.4 g, mean ± sd, respectively). Effect of PFOB on Lung Mechanics Thirty fetuses were subjected to lung function analysis under mechanical ventilation after delivery and completed the whole set of mechanical measurements without any sign of pneumothorax (DH-PFOB, n = 10; DH-saline, n = 10; nonoperated controls, n = 10). Lung mechanics were compared at 30 minutes of ventilation after a steady state for respiratory mechanics was reached (Fig. 1). TLC was significantly lower in DH-saline compared with controls (15.4 ± 7.2 vs 34.5 ± 5.1 μL/g; p < 0.001), whereas instillation of PFOB resulted in a significant increase in TLC when compared with DH-saline (23.4 ± 6.1; p = 0.03) although still significantly lower when compared with nonoperated control animals (p < 0.002). This effect was significantly correlated to LBWR (Pearson correlation, R = 0.75; p = 0.01). Cst was significantly increased in DH-PFOB fetuses compared with DH-saline at every time point of lung function measurement (Fig. 2). Lung recruitment during ventilation had a small effect in DH-saline and control animals (Fig. 2). After 30 minutes, Cst was still increased in DH-PFOB compared with DHsaline (2.38 ± 0.73 vs 1.20 ± 0.43 mL/cm H2O/kg; p < 0.007). Lung tissue and small airway characteristics were not different between DH-PFOB and DH-saline-treated pups. However, all fetuses with DH showed elevated values for Rn, HL, and GL compared with controls (Table 1). mRNA Expression of Surfactant Proteins and the TGF-β Pathway in the Left Lung Normalized mRNA expression levels of surfactant proteins A, B, and C were investigated as indicators of type-II cell function. DH creation and saline treatment resulted in no significant effect on surfactant protein m RNA expression. The levels of surfactant protein mRNAs in DH-PFOB were similar to those in controls and DH-saline (Table 2). Gene expression levels of e382

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Figure 2. Changes in static compliance (Cst) and total lung capacity (TLC) in rabbit fetuses with congenital diaphragmatic hernia (DH) and instillation of perflourooctylbromide (DH-PFOB) or saline (DH-saline). DH-PFOB rabbit pups demonstrated an increase in Cst compared with DH-saline fetuses. TLC was significantly lower in both study groups compared with controls. However, in comparison to DH-saline, the PFOB treatment had a positive effect on TLC compared to nontreated fetuses (*p < 0.05, DH-PFOB as compared to DH-saline, **p < 0.05, controls as compared to DH fetuses, one-way analysis of variance with StudentNeuman-Keuls post hoc correction, error bars indicate 1 sem).

TGF-β isoforms were similar in both DH groups and controls. The same results were observed for the TGF-receptors (Table 2). Effect of PFOB on Extracellular Matrix Formation Genes in the Left Lung We focused on pivotal genes regulating extracellular matrix (ECM) composition (tenascin C [TNC]; lysyloxidase [LOX]; fibulin 5 [FBLN5]; collagen 1 [Col 1]; collagen 3 [Col 3], tropoelastin [T-ELN], and fibrillin-1 [Fib]) as well as ECM remodeling (matrix metalloproteinase-2 [MMP-2] and tissue inhibitor of metalloproteinase [TIMP-1, TIMP-2]). PFOB instillation increased the mRNA expression of fibrillin-1 (p = 0.03) compared with controls (Fig. 3, left lung). Although the level was higher in DH-PFOB compared with DH-saline, the difference was not significant. No differences in mRNA expression were seen in lysyloxidase, fibulin 5, and tropoelastin in both November 2014 • Volume 15 • Number 9

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Table 1. Levels of Newtonian Resistance, Tissue Damping, Tissue Elastance, and Hysteresis in Rabbit Pups With Congenital Diaphragmatic Hernia and Prenatal Instillation of Perfluoroctylbromide or Saline Mean ± sd Forced Oscillation Technique

DH-Perfluoroctylbromide

DH-Saline

Newtonian resistance (cm H2O·s/mL)

0.30 ± 0.11a

0.31 ± 0.14a

Tissue damping (cm H2O/mL)

7.51 ± 3.55

8.18 ± 4.06

Tissue elastance (cm H2O/mL)

34.54 ± 20.78

37.08 ± 26.56

11.60 ± 4.30

0.24 ± 0.04

0.25 ± 0.05

0.28 ± 0.04

Hysteresis

a

Control

0.17 ± 0.06 3.24 ± 1.20

a

a

a

DH = diaphragmatic hernia. a p < 0.01 as compared to controls. The measurements were obtained with the forced oscillation technique (FlexiVent) after 30 min of ventilation in steady-state conditions.

DH-saline and DH-PFOB groups compared to controls and to each other (Fig. 3, left lung). Tenascin mRNA expression tended to be higher in DH-PFOB group compared with controls (p = 0.05). Collagen-1 mRNA expression was up-regulated in DH-PFOB compared with controls (p = 0.01) and DH-saline animals (p = 0.035) (Fig. 3, left lung). However, collagen-3 mRNA expression, although higher in DH-PFOB, was not significantly different when compared with the two other groups. With respect to important regulators of ECM remodeling, PFOB instillation resulted in an increase in metalloproteinase-2, TIMP-1, and TIMP-2 mRNA expression when compared with DH-saline (Fig. 3, left lung). No difference in metalloproteinase-1, metalloproteinase-14, and TIMP-3 expression was observed between the three groups. Effect of PFOB on Gene Expression in the Right Lung As PFOB resulted in significant changes in the mRNA expression of ECM genes in the hypoplastic left lung, we also

investigated their expression in the less hypoplastic right lung. In DH-saline, the right lungs demonstrated an increased mRNA expression of fibrillin-1 and tropoelastin compared with controls (Fig. 3, right lung). PFOB instillation resulted in a decrease in tropoelastin and an increase in metalloproteinase-2 expression compared with DH-saline (Fig. 3, right lung). Compared with controls, TIMP-2 gene expression was significantly decreased in both DH groups. Lung Histology Hypoplastic left lungs instilled with saline demonstrated a disorganized elastin deposition in the lungs compared with controls. The alveoli showed thickened alveolar walls and scattered elastin fibers throughout the walls of distal airways. Almost no elastic fibers were located on the tips of secondary septa (Fig. 4, DH-saline). Intrapulmonary treatment with PFOB resulted in a normal distribution of elastic fibers similar to controls as well as a more defined appearance of the interstitial compartment

Table 2. Relative Messenger RNA Levels of Surfactant Proteins, Transforming Growth Factor-β Isoforms, and Their Receptors in Rabbit Pups Treated With Perfluoroctylbromide or Saline for Congenital Lung Hypoplasia due to Diaphragmatic Hernia Relative Messenger RNA Level (Arbitrary Units), Mean [95% CI] Target Gene

DH-Perfluoroctylbromide

DH-Saline

Controls

Surfactant protein A

0.77 [0.39–1.32]

0.77 [0.51–1.12]

1 [0.65–1.52]

Surfactant protein B

1.31 [0.75–2.63]

1.33 [0.66–2.87]

1 [0.71–1.41]

Surfactant protein C

1.27 [0.76–1.86]

1.41 [0.74–3.33]

1 [0.62–1.62]

TGF-β1

0.90 [0.39–2.10]

0.74 [0.25–2.14]

1 [0.40–2.49]

TGF-β2

1.39 [0.62–3.51]

1.46 [0.54–3.50]

1 [0.52–1.91]

TGF-β3

0.94 [0.53–1.33]

0.97 [0.35–4.78]

1 [0.73–1.37]

TGF rec-1

0.81 [0.42–1.68]

1.05 [0.46–3.81]

1 [0.50–2.00]

TGF rec-2

0.96 [0.40–1.70]

1.32 [0.49–4.99]

1 [0.50–2.01]

DH = diaphragmatic hernia, TGF = transforming growth factor. The messenger RNA levels are obtained with quantitative real-time polymerase chain reaction, normalized to the housekeeping genes 36B4, topoisomerase 1, and succinate dehydrogenase complex, subunit A, and presented in relation to nonoperated controls.

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and TLC (Fig. 2). Although residual PFOB might account for the observed increase in LBWR in DH-PFOB fetuses compared with DH-saline, PFOB remaining in the airways should have been rapidly eliminated during the ventilation period due to its high vapor pressure (34). Muensterer et al (20) have demonstrated that the amount of PFOB remaining in the lungs of prenatally instilled fetuses is minimal even without ventilation (1.56% of the initially instilled volume). In our study, we did not observe any difference in mean LBWR between ventilated and nonventilated DH-PFOB fetuses (0.016 ± 0.003 vs 0.017 ± 0.006). The LBWR in nonventilated and ventilated DH-saline fetuses was also very similar (0.016 ± 0.002, Figure 3. Relative messenger RNA expression of pivotal genes of extracellular matrix formation in the left and not significant compared right lung tissue of fetal rabbit pubs with diaphragmatic hernia (DH) and prenatal perfluorooctylbromide (PFOB) with DH-PFOB nonventilated instillation. Gene expression levels for collagen (Col), fibulin-5 (FBLN5), fibrillin-1 (Fib1), loxyloxidase (LOX), and DH-saline ventilated). tenascin C (TNC), tropoelastin (ELN), metalloproteinases (MMPs), and tissue inhibitor of metalloproteinases (TIMPs) were obtained with quantitative real-time polymerase chain reaction, normalized to the housekeeping Furthermore, Muensterer et al genes 36B4, topoisomerase 1, and succinate dehydrogenase complex, subunit A, and presented in relation (18, 20) have also shown that to nonoperated controls. One-way analysis of variance with Student-Neuman-Keuls correction was used for PFOB instillation increased comparison between surgical groups (DH-PFOB and DH-saline) and controls. DH-PFOB = fetuses with diaphragmatic hernia and prenatal PFOB treatment, DH-saline = diaphragmatic hernia fetuses with fetal saline lung dry weight. In addition, instillation, control = nonoperated control fetuses. (*p < 0.05 DH-PFOB as compared to control, +p < 0.05 when PFOB was used for partial DH-PFOB as compared to DH-saline, **p < 0.05 DH-saline as compared to control.) liquid ventilation postnatally, it has also been shown to increase (Fig. 4, DH-PFOB and control). Quantitative image analy- lung volume in lambs and neonates suffering from DH (14, 23, 35). Thus, taken together, it seems reasonable to speculate that sis confirmed that lung content of elastin was significantly the observed increase in LBWR in DH-PFOB might be due to reduced in DH-saline but not in DH-PFOB animals, where it real lung growth rather than residual PFOB in the lungs. was found to be similar to controls (Fig. 4). The increase in TLC observed in DH-PFOB might reflect an Quantification of trichrome-stained slides showed no difincreased lung volume as a result of enhanced lung growth as lung ference in the amount of collagen fibers between DH and conmorphometry showed normal alveolar architecture and no signs trol rabbit fetuses (data not shown). Distal airspace size, mean of overdistension (Fig. 4 and Table 3). However, even minimal linear intercept, as well as airspace/total tissue fractions were amounts of residual PFOB in the alveoli might have lowered the similar in DH-PFOB, DH-saline, and control groups (Table 3). surface tension in the lungs and thus facilitated the recruitment of lung volume during TLC measurements and lung expansion DISCUSSION during the postmortem fixation process. Many studies have preThis study demonstrates that fetal PFOB treatment of DH viously demonstrated a positive effect of instilled or nebulized improves lung mechanics (Fig. 2) and increases the expres- perfluorocarbon on lung mechanics in different species with or sion of several genes involved in ECM formation and remodelwithout respiratory distress and/or lung hypoplasia (10, 14, 16, ing in the hypoplastic left lung without affecting the mRNA 18, 36, 37). A similar effect could have been present in our model expression of TGF-β subunits, TGF-β receptors, or surfactant (Fig. 2). We can only speculate on whether the increase in TLC proteins (Table 2). In addition, PFOB instillation stimulated in DH-PFOB group is a result of accelerated lung growth or the the process of alveolar formation and normal distribution of physical property of residual PFOB to lower the surface tension elastic fibers compared with DH-saline (Fig. 4). or both. As DH-PFOB fetuses demonstrated high Cst values from In our study, PFOB instillation in fetal rabbit pups with DH the beginning of ventilation with no change over the 30-minute had a positive effect on lung growth as evidenced by LBWR period, it is possible that prenatal PFOB instillation might have e384

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Figure 4. Fetal intratracheal instillation of perfluorooctylbromide (PFOB) restores pulmonary elastin content and pattern in the rabbit model of diaphragmatic hernia (DH). Lung sections stained for elastin showed elastin mainly at septal tips (solid arrowheads) in control fetuses and rabbit pups treated with perfluoroctylbromide (DH-PFOB) prenatally for lung hypoplasia due to DH. To a lesser extent, organized elastin was located in the alveolar walls of controls and DH-PFOB (open arrowheads). Saline-treated fetuses (DH-saline) showed a disorganized elastin pattern with almost no elastin in the secondary crests. Quantitative image analysis confirmed that lung content of elastin, expressed as percentage of lung tissue surface area, was significantly reduced in DH-saline but similar in DH-PFOB as compared to controls. Error bars represent the 95% CI of mean lung elastin content related to total lung tissue (*p < 0.01, analysis of variance with Student-Neuman-Keuls correction).

facilitated alveolar recruitment after birth, and, thus, might have contributed to an increased TLC seen in the DH-PFOB group. We were not able to demonstrate any effect of PFOB on lung tissue forces compared with DH-saline group. GL and HL were significantly increased in both surgical groups compared with controls, reflecting the impact of DH creation on peripheral lung tissue mechanics. As hypothesized in previous studies, this effect could be partly explained by fetal thoracic scarring induced by thoracotomy and subsequent stiffening of the peripheral lung tissue (by pleural adhesions and scarring) in both DH study groups (6, 22). Sustained lung distension induced by TO has been shown to reduce surfactant protein B-positive type-II pneumocyte density (18, 38) and mRNA levels of surfactant protein A and surfactant protein C (39–41). An increase of TGF-β levels has been associated with a decrease in type-II alveolar cells and, thereby, a decrease in surfactant proteins (42). In our study, Pediatric Critical Care Medicine

no change in mRNA expression was observed with respect to the TGF-β subunits, their receptors, or the surfactant proteins (Table 2). The preserved communication between the PFOBdistended hypoplastic lung and surrounding amniotic fluid as well as effective fetal breathing movements may have protected the developing lung against overdistension and induction of the TGF-β pathway with is concomitant problems observed in TO (3). In addition, it is also possible that the anti-inflammatory properties of PFOB might have also contributed to a prevention of induction of the TGF-β pathway, as perfluorocarbon-based treatment modalities have been shown to reduce levels of cytokines, chemokines, and other mediators of pulmonary inflammation (43, 44). PFOB instillation in the hypoplastic left lungs led to upregulation in gene expression of many key proteins involved in ECM formation and remodeling (fibrillin-1, tenascinC, collagen-1, metalloproteinase-2, and TIMP-2). These www.pccmjournal.org

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Table 3. Distal Airspace Size, Mean Chord Length, and Alveolar Area in the Lungs of Diaphragmatic Hernia-Perfluoroctylbromide, Diaphragmatic Hernia-Saline, and Nonoperated Control Fetuses Lung Histology

DH-Perfluoroctylbromide

DH-Saline

Control

Mean chord length

36.1 [33.7; 38.6]

36.8 [32.9; 40.7]

31.2 [26.0; 36.4]

Septal regions (%)

80.8 [75.7; 85.9]

84.9 [82.6; 87.1]

87.2 [79.9; 94.6]

Airspace regions (%)

0.61 [0.58; 0.63]a

0.54 [0.52; 0.55]

0.51 [0.44; 0.57]

Alveolar area (mm2)

6.60 [4.96; 8.24]

6.40 [5.70; 7.10]

6.70 [5.31; 8.08]

DH = diaphragmatic hernia. a p < 0.05 as compared to diaphragmatic hernia-saline and controls, analysis of variance with Student-Newman-Keuls correction.

factors are known for their role in lung maturation (45–48). A concomitant improvement in lung histopathology with increased alveolarization and presence of secondary crests in the DH-PFOB group compared with DH-saline group supports this observation. In addition, the increased gene expression of metalloproteinase-2 in the left lung in DH-PFOB group was accompanied by a concomitant increase in tissue inhibitors of metalloproteinases, which resulted in a MMP-2/TIMP-1 ratio of 1.3 ± 0.6, similar to that observed in DH-saline (1.2 ± 0.5 sd) and controls (1.0 ± 0.5 sd), thereby indicating a physiological balance of ECM turnover. By contrast, TO in DH-rabbit fetuses has been shown to decrease the MMP-2/TIMP-1 ratio due to an increase in TIMP-1 levels after TO (49), thus contradicting the idea that TO might be beneficial for the remodeling of newly formed alveoli. The gene expression of collagen-1 was increased in the left lung in DH-PFOB compared with DH-saline and control (Fig. 3, left lung). However, as the total collagen content assessed by quantitative image analysis was not increased, we presume that the increase in the gene expression of collagen-1 might reflect the physiological induction of lung growth and maturation by fetal PFOB treatment. In our study, one group of operated DH-animals was subjected to intrapulmonary instillation of 0.8 mL of saline (DH-saline). This procedure by itself may have produced some changes in the lungs of the animals. We observed that the left lung hypoplasia in DH-saline animals (LBWR, 0.013 ± 0.003) was not as severe when compared with DH-SHAM animals in our previous study (LBWR, 0.009 ± 0.001) (49). In addition, in our study, no significant difference in tropoelastin expression was observed between DH-saline and control groups (Fig. 3, left lung). Thus, it is possible that instillation of saline also resulted in some changes in the left hypoplastic lungs in our study. However, the effects of PFOB instillation were significantly greater and more physiological as validated by the lung histopathology observations. The effect of PFOB instillation was observed to be different between the more hypoplastic left lung and the less affected right lung. In the right lung, DH-PFOB instillation resulted in a slightly but significantly increased metalloproteinase-2 transcription compared with DH-saline (Fig. 3, right lung). However, the ratio of MMP-2/TIMP-1 was similar to that e386

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observed in the other groups. In addition, no difference in fibrillin-1 and tropoelastin expression was observed compared with controls (Fig. 3, right lung). Some limitations of this study should be acknowledged. The PFOB content in the lungs was not quantified either at birth or at the end of mechanical ventilation. Small amount of PFOB might have been present in the lungs after birth and might have influenced some of the outcome measures due to its ability to lower surface tension. Because of the manual incision in the diaphragm, the size of the diaphragmatic defect and resulting lung hypoplasia is variable. This may account for the wide CIs in gene expression analyses. Yet, despite the variability and small sample size, significant differences between groups were identified. Another limitation of our study is the lack of protein data. We concentrated on gene expression rather than protein expression due to the availability of a limited amount of lung tissue in our model and the lack of validated, nonrabbit-derived antibodies. Various commercially available monoclonal antibodies against collagen and elastin exhibited extensive off-target binding. Therefore, quantitative image analysis was used as a surrogate. However, quantitative protein analysis may provide further insight into the complex turnover of ECM formation and should be evaluated in future animal studies, for example, in the lamb model with comparatively larger lungs. The use of PFOB for fetal lung distension in case of lung hypoplasia in the human fetus might be feasible. However, it has been detected in the fetal brain after intratracheal application in the rabbit fetus (19). The brain tissue concentration was comparable to blood levels determined during postnatal partial liquid ventilation of premature lambs (19, 50). In vitro studies on root ganglion cells have shown an alteration of neuronal cell populations after brief exposure to PFOB (51). Hence, long-term effects of PFOB treatment in neonates cannot be ruled out. The rabbit model of congenital DH used in our study although similar to the human DH in several aspects has its limitations. In contrast to the multifactorial etiology of DH development in human fetuses, DH creation in our model is a sudden one-hit surgical procedure that is created relatively late in gestation by which time pulmonary changes in human DH might have already occurred (52). The rat nitrofen model, on the other hand, is a dual-hit model, consisting of a disturbance in the retinoid and/or thyroid signaling pathways resulting in November 2014 • Volume 15 • Number 9

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DH and subsequent herniation of abdominal viscera into the thorax, mimicking the development of human DH to a great extent (52). Thus, although technically challenging due to the small size of the animal, it would be interesting to study the effect of PFOB instillation in the rat nitrofen model and to determine if PFOB instillation helps in lung growth as well as reversal of pulmonary structural abnormality.

CONCLUSIONS In our study, fetal PFOB instillation in the rabbit model of DH resulted in increased lung growth, stimulation of ECM formation and remodeling, increased lung compliance and, thereby, holds the promise of a new approach in the fetal therapy for lung hypoplasia even though the observed effects might be partly explained by residual PFOB in the lungs. These possible interactions should be focused in further studies to discriminate between the postulated effect of PFOB on lung growth and its physical properties to lower surface tension. The clinical advantage of the self-limiting nature of alveolar distension and stretch due to PFOB instillation and its effect on lung mechanics justifies further evaluation in animal models, for example, the lamb model of DH. A particular focus in further studies could be potential extrapulmonary effects of the PFOB treatment, for example, the direct and indirect effects in the brain and its effects on neuronal cell alteration on gene, protein, and histological level.

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