Impact of congenital diaphragmatic hernia on diaphragm muscle function in neonatal rats

Congenital diaphragmatic hernia (CDH) is characterized by incomplete partitioning of the thoracic and abdominal cavities by the diaphragm muscle (DIAm). The resulting in utero invasion of the abdominal viscera into the thoracic cavity leads to impaired fetal breathing movements, severe pulmonary hypoplasia and pulmonary hypertension. We hypothesized that in a well-established rodent model of nitrofen-induced CDH, DIAm isometric force generation and DIAm fiber cross-sectional areas would be reduced compared to non-lesioned littermate and control pups. In CDH and non-lesioned pups at embryonic day 21 or birth, DIAm isometric force responses to supramaximal field stimulation (200 mA, 0.5 ms duration pulses in 1-s duration trains at rates ranging from 10-100 Hz) was measured ex vivo. Further, DIAm fatigue was determined in response to 120 s of repetitive stimulation at 40 Hz in 330 ms duration trains repeated each second. The DIAm was then stretched to Lo, frozen, and fiber cross-sectional areas were measured in 10 μm transverse sections. In CDH pups, there was a marked reduction in DIAm specific force and force following 120 s of fatiguing contraction. The cross-sectional area of DIAm fibers was also reduced in CDH pups compared to non-lesioned littermates and control pups. These results show that CDH is associated with a dramatic weakening of the DIAm, which may contribute to poor survival despite various surgical efforts to repair the hernia and improve lung development.

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Effect of nitrofen in the final stages of development of the diaphragm muscle in rats

Acta Cirurgica Brasileira ◽

10.1590/s0102-86502013001300004 ◽

2013 ◽

Vol 28 (suppl 1) ◽

pp. 13-18 ◽

Cited By ~ 1

Author(s):

Frances Lilian Lanhellas Gonçalves ◽

Fábio Santana de Oliveira ◽

Augusto Frederico Schmidt ◽

Luís Antônio Violin Dias Pereira ◽

Rodrigo Melo Gallindo ◽

...

Keyword(s):

Body Weight ◽

Congenital Diaphragmatic Hernia ◽

Olive Oil ◽

Diaphragmatic Hernia ◽

Muscle Fibers ◽

Diaphragm Muscle ◽

External Control ◽

Stages Of Development ◽

Pregnant Rats ◽

Histological Staining

PURPOSE: To evaluate the expression of myosin in muscle fibers of the diaphragm in experimental congenital diaphragmatic hernia (CDH). METHODS: Fetuses of pregnant rats were divided into four groups: External Control (EC), composed of non-manipulated rats; Nitrofen, composed of pregnant rats that received 100 mg of nitrofen (2,4-dichloro-4'nitrodiphenyl ether) diluted in olive oil on gestational day (GD) 9.5, whose fetuses developed CDH (N+) or not (N-), and Olive Oil Placebo (OO), composed of pregnant rats that received the oil on the same GD. The fetuses were collected on GD 18.5, 19.5, 20.5 and 21.5 (term = 22 days). We obtained body weight (BW) and photographed the diaphragm area (DA), hernia area (HA) and subsequent calculated the HA/DA ratio in N+ group. Samples of Diaphragm muscle were processed for histological staining with H/E and immunohistochemistry (IHQ) for myosin.} RESULTS: The fetuses of N- and N+ groups had decreased BW and DA compared to EC and OO groups (p <0.001). HA was decreased on GD 18.5 compared to 21.5 (p <0.001) and the HA/DA ratio showed no difference. IHQ showed decreased expression of myosin in nitrofen groups. CONCLUSION: CDH induced by nitrofen model contributes to the understanding of muscularization in the formation of the diaphragm where the myosin expression is decreased.

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Prenatal administration of neuropeptide bombesin promotes lung development in a rat model of nitrofen-induced congenital diaphragmatic hernia

Journal of Pediatric Surgery ◽

10.1016/j.jpedsurg.2014.09.015 ◽

2014 ◽

Vol 49 (12) ◽

pp. 1749-1752 ◽

Cited By ~ 7

Author(s):

Kohei Sakai ◽

Osamu Kimura ◽

Taizo Furukawa ◽

Shigehisa Fumino ◽

Koji Higuchi ◽

...

Keyword(s):

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Rat Model ◽

Lung Development

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Thyroid transcription factor-1 expression during normal human lung development and in patients with congenital diaphragmatic hernia

Journal of Pediatric Surgery ◽

10.1053/jpsu.2002.34977 ◽

2002 ◽

Vol 37 (9) ◽

pp. 1258-1262 ◽

Cited By ~ 9

Author(s):

M. Hösgör ◽

Y. IJzendoorn ◽

W.J. Mooi ◽

D. Tibboel ◽

R.R. de Krijger

Keyword(s):

Transcription Factor ◽

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Lung Development ◽

Human Lung ◽

Thyroid Transcription Factor 1 ◽

Thyroid Transcription Factor ◽

Normal Human ◽

Transcription Factor 1

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Amniotic fluid stem cell extracellular vesicles promote fetal lung branching and cell differentiation in experimental congenital diaphragmatic hernia

10.1101/2022.01.10.475632 ◽

2022 ◽

Author(s):

Kasra Khalaj ◽

Rebeca Lopes Figueira ◽

Lina Antounians ◽

Sree Gandhi ◽

Matthew Wales ◽

...

Keyword(s):

Amniotic Fluid ◽

Congenital Diaphragmatic Hernia ◽

Extracellular Vesicles ◽

Diaphragmatic Hernia ◽

Lung Development ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Neuroendocrine Cells ◽

Beta Catenin

Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired branching morphogenesis and differentiation. We have previously demonstrated that administration of extracellular vesicles derived from rat amniotic fluid stem cells (AFSC-EVs) rescues development of hypoplastic lungs at the pseudoglandular and alveolar stages in rodent models of CDH. Herein, we tested whether AFSC-EVs exert their regenerative effects at the canalicular and saccular stages, as these are translationally relevant for clinical intervention. To induce fetal pulmonary hypoplasia, we gavaged rat dams with nitrofen at embryonic day 9.5 and demonstrated that nitrofen-exposed lungs had impaired branching morphogenesis, dysregulated signaling pathways relevant to lung development (FGF10/FGFR2, ROBO/SLIT, Ephrin, Neuropilin 1, beta-catenin) and impaired epithelial and mesenchymal cell marker expression at both stages. AFSC-EVs administered to nitrofen-exposed lung explants rescued airspace density and increased the expression levels of key factors responsible for branching morphogenesis. Moreover, AFSC-EVs rescued the expression of alveolar type 1 and 2 cell markers at both canalicular and saccular stages, and restored markers of club, ciliated epithelial, and pulmonary neuroendocrine cells at the saccular stage. AFSC-EV treated lungs also had restored markers of lipofibroblasts and PDGFRA+ cells to control levels at both stages. EV tracking showed uptake of AFSC-EV RNA cargo throughout the fetal lung and an mRNA-miRNA network analysis identified that several miRNAs responsible for regulating lung development processes were contained in the AFSC-EV cargo. These findings suggest that AFSC-EV based therapies hold potential for restoring fetal lung growth and maturation in babies with pulmonary hypoplasia secondary to CDH.

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miR-200 family expression during normal and abnormal lung development due to congenital diaphragmatic hernia at the later embryonic stage in the nitrofen rat model

Pediatric Surgery International ◽

10.1007/s00383-020-04757-2 ◽

2020 ◽

Vol 36 (12) ◽

pp. 1429-1436

Author(s):

Drew Mulhall ◽

Naghmeh Khoshgoo ◽

Robin Visser ◽

Barbara Iwasiow ◽

Chelsea Day ◽

...

Keyword(s):

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Rat Model ◽

Lung Development ◽

Embryonic Stage

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Pathogenesis of nitrofen-induced congenital diaphragmatic hernia in fetal rats

Journal of Applied Physiology ◽

10.1152/jappl.1997.83.2.338 ◽

1997 ◽

Vol 83 (2) ◽

pp. 338-347 ◽

Cited By ~ 86

Author(s):

Douglas W. Allan ◽

John J. Greer

Keyword(s):

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Rat Model ◽

Phrenic Nerve ◽

Lung Development ◽

Primary Role ◽

Primary Defect ◽

Myotube Formation ◽

Fetal Rats ◽

Weight Protein

Allan, Douglas W., and John J. Greer. Pathogenesis of nitrofen-induced congenital diaphragmatic hernia in fetal rats. J. Appl. Physiol. 83(2): 338–347, 1997.—Congenital diaphragmatic hernia (CDH) is a developmental anomaly characterized by the malformation of the diaphragm and impaired lung development. In the present study, we tested several hypotheses regarding the pathogenesis of CDH, including those suggesting that the primary defect is due to abnormal 1) lung development, 2) phrenic nerve formation, 3) developmental processes underlying diaphragmatic myotube formation, 4) pleuroperitoneal canal closure, or 5) formation of the primordial diaphragm within the pleuroperitoneal fold. The 2,4-dichloro-phenyl- p-nitrophenyl ether (nitrofen)-induced CDH rat model was used for this study. The following parameters were compared between normal and herniated fetal rats at various stages of development: 1) weight, protein, and DNA content of lungs; 2) phrenic nerve diameter, axonal number, and motoneuron distribution; 3) formation of the phrenic nerve intramuscular branching pattern and diaphragmatic myotube formation; and 4) formation of the precursor of the diaphragmatic musculature, the pleuroperitoneal fold. We demonstrated that previously proposed theories regarding the primary role of the lung, phrenic nerve, myotube formation, and the closure of pleuroperitoneal canal in the pathogenesis of CDH are incorrect. Rather, the primary defect associated with CDH, at least in the nitrofen rat model, occurs at the earliest stage of diaphragm development, the formation of the pleuroperitoneal fold.

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