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