Pediatric/Congenital/Developmental
Heterogeneous Response in Rabbit Fetal Diaphragmatic Hernia Lungs After Tracheal Occlusion

https://doi.org/10.1016/j.jss.2019.12.025Get rights and content

Abstract

Background

Fetal tracheal occlusion (TO) is an experimental therapeutic approach to stimulate lung growth in the most severe congenital diaphragmatic hernia (CDH) cases. We have previously demonstrated a heterogeneous response of normal fetal rabbit lungs after TO with the appearance of at least two distinct zones. The aim of this study was to examine the fetal lung response after TO in a left CDH fetal rabbit model.

Methods

Fetal rabbits at 25 d gestation underwent surgical creation of CDH followed by TO at 27 d and harvest on day 30. Morphometric analysis, global metabolomics, and fluorescence lifetime imaging microscopy (FLIM) were performed to evaluate structural and metabolic changes in control, CDH, and CDH + TO lungs.

Results

Right and left lungs were different at the baseline and had a heterogeneous pulmonary growth response in CDH and after TO. The relative percent growth of the right lungs in CDH + TO was higher than the left lungs. Morphometric analyses revealed heterogeneous tissue-to-airspace ratios, in addition to size and number of airspaces within and between the lungs in the different groups. Global metabolomics demonstrated a slower rate of metabolism in the CDH group with the left lungs being less metabolically active. TO stimulated metabolic activity in both lungs to different degrees. FLIM analysis demonstrated local heterogeneity in glycolysis, oxidative phosphorylation (OXPHOS), and FLIM “lipid-surfactant” signal within and between the right and left lungs in all groups.

Conclusions

We demonstrate that TO leads to a heterogeneous morphologic and metabolic response within and between the right and left lungs in a left CDH rabbit model.

Introduction

Congenital diaphragmatic hernia (CDH) is one of the most common and devastating conditions known to pediatric surgeons. Worldwide, a baby with CDH is born every 10 min.1 The estimated annual cost of CDH in the United States alone is more than $230 million.2 Although most cases of CDH are surgically correctable, these patients suffer significant morbidities including pulmonary hypertension in the neonatal period and long-term ventilatory insufficiency due to pulmonary hypoplasia and abnormal lung development.3, 4, 5 Furthermore, despite significant advancements in supportive neonatal critical care, mortality in the most severe CDH cases is unacceptably high.6, 7, 8, 9 Thus, interventions to improve the outcomes of these patients must focus on fetal intervention.

The rationale for fetal therapy in severe CDH is to improve in utero fetal lung growth for neonatal survival. Experimental fetal tracheal occlusion (TO) is the only available therapeutic intervention for CDH besides neonatal supportive care. TO obstructs the fetal trachea and interrupts the normal egress of lung fluid during pulmonary development leading to increased levels of mechanical tissue stretch and growth factors, resulting in increased cell proliferation and accelerated lung growth. We recently published the first study to describe the proteome of tracheal fluid in a large animal model of CDH with and without fetal TO.10 Proteomic profiling of the tracheal fluid from fetal sheep with CDH and TO validates finding in other models with regards to the induction of cellular proliferation11, 12, 13 and suggests a role for RAC-alpha serine/threonine-protein kinase (AKT, also known as protein kinase B) in this process.

Morbidity and mortality in CDH are the result of the complex interplay between pulmonary hypoplasia, pulmonary hypertension, and cardiac dysfunction. Hypoplastic lungs are characterized by the reduced surface area for gas exchange manifested by decreased number of alveoli and alveolar simplification.14 Fetal lung fluid production augmented by fetal breathing movements is by far the most important biophysical factor responsible for mechanotransduction.15 Fetal lung fluid is formed by the alveolar epithelial cells of the distal airways and gradually increases toward late gestation.16 Occluding the trachea leads to fluid accumulation in the lung.

After TO, the developing lung is exposed to supraphysiologic levels of mechanical stretch. Although this induces lung growth, underlying alveolar development and function is abnormal.17,18 We have previously published that, when assessed histologically, areas of the occluded lungs demonstrate acini with control-like small airspaces and different distribution of alveolar and airspace sizes interspersed among areas with enlarged airspaces.

In addition, we have described a differential metabolic and morphometric response that is dependent on the duration of TO (day 1 versus day 4 lungs.) One day after TO, we observed normal growth of airspaces with increased oxidative phosphorylation (OXPHOS) and normal fluorescence lifetime imaging microscopy (FLIM) “lipid-surfactant” signal in some areas. However, after 4 d, there is formation of two distinct areas with some areas demonstrating enlarged airspaces and increased glycolysis with decreased FLIM “lipid-surfactant” signal interspersed among control-like areas.19 This finding may highlight the perturbed fuel utilization after TO.20 Given that rapid cellular growth and proliferation need large amounts of building blocks including amino acids and nucleotides, cells preparing for proliferation use a unique repertoire of metabolic processes with a shift to a “Warburg-like metabolism,” which is dependent on aerobic glycolysis.21 Both the tricarboxylic acid cycle (TCA) and glycolysis play crucial roles in adenosine triphosphate (ATP) production, but their speeds and efficiencies are different.22 Metabolic flexibility is very important for adaptation to different microenvironmental conditions.23 Differentiated cells mostly use OXPHOS to efficiently generate necessary amounts of ATP,21,24 whereas rapidly proliferating cells and cells devoid of adequate oxygen shift to increased nutrient uptake and glycolysis.21

The metabolic landscape after TO in the left CDH rabbit model is currently unknown. Furthermore, it is unknown if the left CDH rabbit model will be accompanied by heterogeneity of the distal pulmonary acinar morphometry and/or variability between the right and left lungs. We hypothesize that there will be a variable parenchymal response in CDH lungs subjected to TO in fetal rabbits. In this study, we investigate the local morphometric and metabolic changes accompanying left CDH in the fetal rabbit model and examine the changes in these parameters after TO.

Section snippets

Fetal rabbit tracheal occlusion model

All animal procedures were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC), Cincinnati Children's Hospital Medical Center (IACUC # 2013-0294). Time-dated pregnant New Zealand white rabbits were obtained from the Charles River Laboratory and housed at 24°C in separate cages under standard laboratory conditions with free access to water and chow.

Experimental groups

Pregnant

Heterogeneous pulmonary growth response between left and right lungs in the left CDH model after TO

It is well known that CDH is characterized by a decreased LBWR compared with unaffected subjects.1,30,31 TO has been shown to induce an accelerated lung growth in controls and CDH cases.19,28,31, 32, 33 Hence, an augmented total LBWR (TLBWR) is expected after TO. Consistently, the left CDH fetuses demonstrated a statistically significant decrease in TLBWR compared with control animals (0.012 ± 0.001 and 0.022 ± 0.001, respectively, P < 0.0001.) TO corrected the deficit in the TLBWR compared to

Discussion

In this article, we investigated the pulmonary morphometric and metabolic changes in response to TO in the fetal rabbit left CDH model. We observed heterogeneity within and between left and right lungs after TO demonstrated by relative percent changes in the LBWR, TAR distributions, average size of airspaces, and metabolic indices. The reasons for these structural and metabolic variabilities are currently unknown, constituting a critical barrier to scientific progress and development of novel

Conclusions

Commensurate with our previous findings in normal fetal rabbit lungs, the experiments outlined in this study demonstrate conservation of the heterogeneous response after TO in CDH. Specifically, TO leads to a heterogenous morphologic and metabolic response in the fetal rabbit CDH model. Future experiments are needed to examine the functional effects of these changes using the flexiVent system to measure the respiratory mechanics of the lungs from newborn rabbits and after maintenance under

Acknowledgment

The authors would like to thank the University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core.

Authors contribution: Evgenia Dobrinskikh conceptualized the study, performed the histological staining and imaging, FLIM imaging, and analyzed and interpreted the data. Saif I. Al-Juboori conceptualized the study, performed histological analyses, histograms fitting and their statistical comparisons, and analyzed and interpreted the data. Marc Oria and Jose L. Peiro conceptualized

References (43)

  • E. Hannezo et al.

    A unifying theory of branching morphogenesis

    Cell

    (2017)
  • L.R. Putnam et al.

    Congenital diaphragmatic hernia defect size and infant morbidity at discharge

    Pediatrics

    (2016)
  • J. Balayla et al.

    Incidence, predictors and outcomes of congenital diaphragmatic hernia: a population-based study of 32 million births in the United States

    J Matern Fetal Neonatal Med

    (2014)
  • R. Ruano et al.

    A randomized controlled trial of fetal endoscopic tracheal occlusion versus postnatal management of severe isolated congenital diaphragmatic hernia

    Ultrasound Obstet Gynecol

    (2012)
  • J.C. Jani et al.

    Severe diaphragmatic hernia treated by fetal endoscopic tracheal occlusion

    Ultrasound Obstet Gynecol

    (2009)
  • J.L. Peiro et al.

    Proteomic profiling of tracheal fluid in an ovine model of congenital diaphragmatic hernia and fetal tracheal occlusion

    Am J Physiol Lung Cell Mol Physiol

    (2018)
  • A.C. Engels et al.

    Pulmonary transcriptome analysis in the surgically induced rabbit model of diaphragmatic hernia treated with fetal tracheal occlusion

    Dis Model Mech

    (2016)
  • R. Ruano et al.

    Fetal pulmonary response after fetoscopic tracheal occlusion for severe isolated congenital diaphragmatic hernia

    Obstet Gynecol

    (2012)
  • T. Seaborn et al.

    Identification of cellular processes that are rapidly modulated in response to tracheal occlusion within mice lungs

    Pediatr Res

    (2008)
  • O. Boucherat et al.

    Decreased lung fibroblast growth factor 18 and elastin in human congenital diaphragmatic hernia and animal models

    Am J Respir Crit Care Med

    (2007)
  • P.A. Khan et al.

    Tracheal occlusion: a review of obstructing fetal lungs to make them grow and mature

    Am J Med Genet C Semin Med Genet

    (2007)
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