(H-294) Pulmonary Vascular Changes Consistent with Pulmonary Hypertension are Present in a Nitrofen/Bisdiamine Murine Model of Congenital Diaphragmatic Hernia

Introduction:: Congenital diaphragmatic hernia (CDH) is a severe birth defect that affects approximately 2.3 out of every 10,000 live births¹. It occurs when the diaphragm fails to fully develop during gestation, allowing abdominal organs such as the stomach, liver, and intestines to protrude into the chest cavity, thereby compressing the lungs. CDH most commonly results in pulmonary hypoplasia and pulmonary hypertension (PH). Despite its severity, many aspects of CDH, including its etiology, remain unknown. Various animal models have been created to gain insights into the underlying pathology of CDH. One such model is the nitrofen and bisdiamine (N/B) mouse model, where these drugs, administered orally to pregnant mice, inhibit the retinoic acid pathway, leading to diaphragmatic hernias and pulmonary hypoplasia. Although this model has successfully replicated one key feature of human CDH, hypoplasia, it has yet to be thoroughly examined to determine if it also leads to a PH-like phenotype. Thus, our objective was to investigate the vascular changes associated with CDH-PH in the N/B mouse model.


Materials and Methods:: Pregnant CD1 mice were dosed with nitrofen (0.5 g/kg bw) and bisdiamine (0.125 g/kg bw) or extra virgin olive oil (EVOO) as control at embryonic day (E) 8.5. At E18.5, mice were sacrificed, and each embryo and its respective lungs were dissected. Embryonic lungs were fixed with 4% paraformaldehyde and either 1) dehydrated in 70% ethanol, processed and paraffin-embedded and sectioned for histology, or 2) cryoprotected in sucrose, frozen in optimal cutting temperature compound using liquid nitrogen and cryosectioned for immunofluorescent (IF) staining. Histological sections were stained with hematoxylin and Masson’s Trichrome to measure collagen deposition and localization and to measure vascular lumen diameter. For IF imaging, tissues were permeabilized, blocked, and stained for PECAM-1, endomucin, alpha-smooth muscle actin, and DAPI and were imaged using confocal microscopy. Smooth muscle hypertrophy around the vessel walls was measured via the thickness of the alpha-smooth muscle stain.


Results, Conclusions, and Discussions:: Diagnosing pulmonary hypertension (PH) in humans involves direct pressure measurement via catheterization, however, this is not possible in fetal and neonatal mice. Therefore, we focused on examining morphological changes in the vasculature associated with CDH-PH including changes in arterial lumen diameter, smooth muscle hypertrophy, and extracellular matrix (ECM) deposition. At E18.5, vessels in embryonic lungs from N/B-dosed animals had decreased lumen area (H&E) and increased smooth muscle thickness (IF) around arteriole walls when compared to EVOO controls. Additionally, there was increased collagen deposition throughout the lung parenchyma and perivascular regions in the N/B mouse model relative to EVOO controls.

The results of our study indicate that the N/B CDH mouse model accurately reproduces a phenotype similar to human CDH-PH. This study represents the initial examination of vascular alterations associated with this specific CDH mouse model, allowing for further exploration of CDH etiology and progression in relation to both pulmonary hypoplasia and pulmonary hypertension.


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References (Optional): : [1] Politis, Ann Epidemiol. 2021.