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The mammalian pulmonary vasculature consists of functionally and morphologically heterogeneous compartments. When comparing sets of lungs, for example, in disease models or therapeutic interventions, local changes may be masked by the overall heterogeneity of the organ structure. Therefore, alterations taking place only in a sub-compartment may not be detectable by global analysis. In the monopodial lung, the characterization of distinct vessel groups is difficult, due to the asymmetrical branching pattern. In this pilot study, a previously established method to classify segments of the monopodial pulmonary arterial tree into homogeneous groups was employed. To test its suitability for experimental settings, the method was applied to a hyperoxia (HYX, ≥95% oxygen) rabbit model of bronchopulmonary dysplasia and a normoxic control group (NOX, 21% oxygen). The method allowed the identification of morphological differences between the HYX and the NOX groups. Globally visible differences in lumen diameter were pinpointed to specific lung regions. Furthermore, local changes of wall dimension and cell layers in single compartments, that would not have been identifiable in an unfocused analysis of the whole dataset, were found. In conclusion, the described method achieves a higher precision in morphological studies of lung disease models, compared to a common, global analysis approach., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Bronchopulmonary dysplasia (BPD) is a developmental disorder of infants born prematurely, characterized by disrupted alveolarization and microvascular maturation. However, the sequence of alveolar and vascular alterations is currently not fully understood. Therefore, we used a rabbit model to evaluate alveolar and vascular development under preterm birth and hyperoxia, respectively. Pups were born by cesarean section 3 days before term and exposed for 7 days to hyperoxia (95% O 2 ) or normoxia (21% O 2 ). In addition, term-born rabbits were exposed to normoxia for 4 days. Rabbit lungs were fixed by vascular perfusion and prepared for stereological analysis. Normoxic preterm rabbits had a significantly lower number of alveoli than term rabbits. The number of septal capillaries was lower in preterm rabbits but less pronounced than the alveolar reduction. In hyperoxic preterm rabbits, the number of alveoli was similar to that in normoxic preterm animals; however, hyperoxia had a severe additional negative effect on the capillary number. In conclusion, preterm birth had a strong effect on alveolar development, and hyperoxia had a more pronounced effect on capillary development. The data provide a complex picture of the vascular hypothesis of BPD which rather seems to reflect the ambient oxygen concentration than the effect of premature birth.

Ventilation and respiratory care have substantially changed over the last decades in extremely premature neonates but the impact on respiratory health remains largely unclear. To determine changes in respiratory care and disease frequency in extremely premature infants, a retrospective single-centre cohort study of extremely preterm infants was performed. All infants born alive between 24 + 0 and 27 + 6 weeks of gestation in 2000-2001 (Epoch 1), 2009-2010 (Epoch 2), and 2018-2019 (Epoch 3) were included. The primary outcome of this study was the incidence of bronchopulmonary dysplasia (BPD, diagnosed according to three different criteria) or death. Secondary outcomes included the usage of different ventilation modes, changes in pharmacotherapy, and the incidence of significant extra-pulmonary morbidities. A total of 184 neonates were included of whom 151 survived until 36 weeks of corrected GA (cGA). Oxygen or positive pressure dependence increased over time (26.1%, 41.7%, and 56.1% respectively), with higher adjusted odds in Epoch 3 for the composite outcome "BPD or death" (adjusted odds ratio: 2.55 [95%CI 1.19-5.61]). Severity-based definitions showed increasing trends in survivors only. Time spent on invasive mechanical ventilation was similar throughout the years, but the use of non-invasive ventilation significantly increased in Epoch 3 (32.0 [95%CI 25.0-37.0] vs 27.0 [95%CI 26.0-32.0] vs 53.0 [95%CI 46.0-58.0] days). Moreover, mortality-adjusted rates of severe IVH, NEC, or intestinal perforation and multiple sepsis tended to decrease.   Conclusion: In spite of significant clinical advancements and adherence to novel treatment guidelines in our neonatal intensive care unit, the incidence of BPD increased over time. What is Known: • Rates of BPD are stable or increase in population-based studies. • Extremely preterm infants are particularly susceptible to developing BPD. What is New: • Despite increased use of evidence-based corticosteroid administration and early initiation of caffeine, the incidence of BPD has not decreased over the past decade.  • Increased usage of non-invasive ventilation is associated with an increase of BPD incidence., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Reproducible and unbiased methods to quantify alveolar structure are important for research on many lung diseases. However, manually estimating alveolar structure through stereology is time consuming and inter-observer variability is high. The objective of this work was to develop and validate a fast, reproducible and accurate (semi-)automatic alternative. A FIJI-macro was designed that automatically segments lung images to binary masks, and counts the number of test points falling on tissue and the number of intersections of the air-tissue interface with a set of test lines. Manual selection remains necessary for the recognition of non-parenchymal tissue and alveolar exudates. Volume density of alveolar septa ([Formula: see text]) and mean linear intercept of the airspaces (Lm) as measured by the macro were compared to theoretical values for 11 artificial test images and to manually counted values for 17 lungs slides using linear regression and Bland-Altman plots. Inter-observer agreement between 3 observers, measuring 8 lungs both manually and automatically, was assessed using intraclass correlation coefficients (ICC). [Formula: see text] and Lm measured by the macro closely approached theoretical values for artificial test images (R2 of 0.9750 and 0.9573 and bias of 0.34% and 8.7%). The macro data in lungs were slightly higher for [Formula: see text] and slightly lower for Lm in comparison to manually counted values (R2 of 0.8262 and 0.8288 and bias of -6.0% and 12.1%). Visually, semi-automatic segmentation was accurate. Most importantly, manually counted [Formula: see text] and Lm had only moderate to good inter-observer agreement (ICC 0.859 and 0.643), but agreements were excellent for semi-automatically counted values (ICC 0.956 and 0.900). This semi-automatic method provides accurate and highly reproducible alveolar morphometry results. Future efforts should focus on refining methods for automatic detection of non-parenchymal tissue or exudates, and for assessment of lung structure on 3D reconstructions of lungs scanned with microCT., Competing Interests: The authors have declared that no competing interests exist.

Background: Bronchopulmonary dysplasia (BPD) remains a frequent complication following preterm birth, affecting respiratory health throughout life. Transcriptome analysis in a preterm rabbit model for BPD revealed dysregulation of key genes for inflammation, vascular growth and lung development in animals exposed to hyperoxia, which could be prevented by simvastatin., Methods: Preterm rabbits were randomized to either normoxia (21% O 2 ) or hyperoxia (95% O 2 ) and within each condition to treatment with 5 mg/kg simvastatin daily or control. Lung function, structure and mRNA-expression was assessed on day 7., Results: Simvastatin partially prevented the effect of hyperoxia on lung function, without altering alveolar structure or inflammation. A trend towards a less fibrotic phenotype was noted in simvastatin-treated pups, and airways were less muscularized. Most importantly, simvastatin completely prevented hyperoxia-induced arterial remodeling, in association with partial restoration of VEGFA and VEGF receptor 2 (VEGFR2) expression. Simvastatin however decreased survival in pups exposed to normoxia, but not to hyperoxia., Conclusion: Repurposing of simvastatin could be an advantageous therapeutic strategy for bronchopulmonary dysplasia and other developmental lung diseases with pulmonary vascular disease. The increased mortality in the treated normoxia group however limits the translational value at this dose and administration route.

A significant proportion of preterm infants develop bronchopulmonary dysplasia (BPD) leading to poor lifelong respiratory health. Limited treatment options exist with continuous positive airway pressure (CPAP) ventilation being one of the few associated with diminished BPD. However, little is known about the effect of the distending pressure of CPAP on the developing lung exposed to hyperoxia. We aimed to identify the functional and structural effects of CPAP in a preterm hyperoxia rabbit model of BPD. Premature rabbit pups were randomized to normoxia, hyperoxia (≥95% O 2 ), or hyperoxia plus 4 h daily CPAP [fraction of inspired oxygen (Fi O 2 ) 0.95, 5 cmH 2 O]. On day 7 postdelivery we performed invasive pressure-volume- and forced oscillation-based pulmonary function tests, before lung harvest for histological evaluation. Alveolar and vascular morphology, airway smooth muscle content, respiratory epithelium height, extracellular matrix components, and inflammatory cytokine expression were quantified. Hyperoxia-reared pups had restrictive lungs: alveolar walls were thickened, with the lung parenchymal tissue, collagen content, and airway smooth muscle content increased. In addition, peripheral pulmonary artery wall thickness was increased. CPAP increased alveolar recruitment and limited the structural effect of hyperoxia on the respiratory epithelium and pulmonary arteries. Additionally, CPAP improved lung function, mitigating hyperoxia-associated changes to respiratory system resistance, tissue damping, and tissue elastance. Hyperoxia disrupted functional and structural lung development. Daily intermittent CPAP limited hyperoxia-associated decreased lung function and attenuated structural changes to pulmonary arteries and respiratory epithelium while having no structural alveolar consequences. The mechanism by which CPAP has these beneficial effects needs further investigation.

Recent clinical trials in newborns have successfully used surfactant as a drug carrier for an active compound, to minimize systemic exposure. To investigate the translational potential of surfactant-compound mixtures and other local therapeutics, a relevant animal model is required in which intratracheal administration for maximal local deposition is technically possible and well tolerated. Preterm rabbit pups (born at 28 days of gestation) were exposed to either hyperoxia or normoxia and randomized to receive daily intratracheal surfactant, daily intratracheal saline, or no injections for 7 days. At day 7, the overall lung function and morphology were assessed. Efficacy in terms of distribution was assessed by micro-PET-CT on both day 0 and day 7. Lung function as well as parenchymal and vascular structure were altered by hyperoxia, thereby reproducing a phenotype reminiscent of bronchopulmonary dysplasia (BPD). Neither intratracheal surfactant nor saline affected the survival or the hyperoxia-induced BPD phenotype of the pups. Using PET-CT, we demonstrate that 82.5% of the injected radioactive tracer goes and remains in the lungs, with a decrease of only 4% after 150 min. Surfactant and saline can safely and effectively be administered in spontaneously breathing preterm rabbits. The described model and method enable researchers to evaluate intratracheal pharmacological interventions for the treatment of BPD.

Bronchopulmonary dysplasia (BPD) remains the most common complication of extreme prematurity as no effective treatment is available to date. This calls for the exploration of new therapeutic options like cell therapy, which is already effective for various human (lung) disorders. We systematically searched the MEDLINE, Embase, and Web of Science databases from the earliest date till January 2017 and included original studies on the perinatal use of cell-based therapies (i.e. cells and/or cell-derivatives) to treat BDP in animal models. Fourth publications describing 47 interventions were retrieved. Newborn mice/rats raised in a hyperoxic environment were studied in most interventions. Different cell types - either intact cells or their conditioned medium - were administered, but bone marrow and umbilical cord blood derived mesenchymal stem cells were most prevalent. All studies reported positive effects on outcome parameters including alveolar and vascular morphometry, lung function, and inflammation. Cell homing to the lungs was demonstrated in some studies, but the therapeutic effects seemed to be mostly mediated via paracrine modulation of inflammation, fibrosis and angiogenesis., Conclusion: Multiple rat/mouse studies show promise for cell therapy for BPD. Yet careful study of action mechanisms and side effects in large animal models is imperative before clinical translation can be achieved.

Background: Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects extremely preterm infants and remains - despite improvements in neonatal intensive care - a major cause of neonatal mortality and morbidity. Cell-therapeutic strategies employing mesenchymal stem cells (MSC) have been shown to modulate lung development in BPD models., Objective: Herein, we evaluate the potential of human amniotic fluid (hAF)-SC and hAF-SC with upregulated expression of vascular endothelial growth factor (VEGF) as cell-therapeutic agents for BPD., Methods: Preterm rabbit pups were raised in normoxia (21% O2) or hyperoxia (≥95% O2). Hyperoxia-exposed pups randomly received an intraperitoneal injection of fibroblasts, naïve hAF-SC, or hAF-SC-VEGF on postnatal day (PN) 0. On PN7, surviving pups were tested for pulmonary (forced oscillation technique) and vascular (pulmonary artery Doppler ultrasound) function, and lungs were processed for morphometric measurements of parenchymal and vascular structure and inflammation., Results: Intraperitoneal injection of cells resulted in homing to the lungs. The lungs of hyperoxia-exposed animals displayed parenchymal and vascular structural and functional damage reminiscent of BPD, which was significantly improved after treatment with hAF-SC-VEGF. Treating hyperoxia-exposed animals with naïve AF-SC attenuated only the lung inflammation and the vascular structural defect. Treatment with fibroblasts, which were used as a cellular control, did not lead to any improvements., Conclusion: hAF-SC with upregulated VEGF expression display enhanced potential to prevent/reverse lung injury in preterm rabbits, whereas naïve hAF-SC only show a moderate therapeutic potential. These results point towards an added value of VEGF delivered by hAF-SC in the treatment of BPD., (© 2018 S. Karger AG, Basel.)

Bronchopulmonary dysplasia (BPD) remains a frequent and disabling consequence of preterm birth, despite the recent advances in neonatal intensive care. There is a need to further improve outcomes and many novel therapeutic or preventive strategies are therefore investigated in animal models. We discuss in this review the aspects of human BPD pathophysiology and phenotype, which ideally should be mimicked by an animal model for this disease. Prematurity remains the common denominator in the heterogeneous spectrum of human BPD, and preterm animal models thus have a clear translational advantage. Additional factors, like excessive oxygen, mechanical ventilation and infection, which frequently have been studied in animal models, can contribute to preterm lung injury however are not indispensable to develop BPD. The phenotype of human BPD is characterized by alveolar developmental arrest with extracellular matrix remodeling, signs of obstructive airway disease and pulmonary vascular disease. Many animal models mimic this phenotype and have their place in BPD research, but results should be interpreted bearing in mind the specific advantages and disadvantages of the model. Term mice and rats are well suited for basic explorative research on specific disease mechanisms, essential for the generation of new hypotheses, while the larger ventilated preterm baboons and lambs provide a good platform for the ultimate translation of these strategies towards clinical application. The preterm rabbit model seems a promising model as it the smallest model that includes a factor of prematurity and has a unique position between the small and large animal models., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Bronchopulmonary dysplasia (BPD) is caused by preterm neonatal lung injury and results in oxygen dependency and pulmonary hypertension. Current clinical management fails to reduce the incidence of BPD, which calls for novel therapies. Fetal rabbits have a lung development that mimics humans and can be used as a translational model to test novel treatment options. In preterm rabbits, exposure to hyperoxia leads to parenchymal changes, yet vascular damage has not been studied in this model. In this study we document the early functional and structural changes of the lung vasculature in preterm rabbits that are induced by hyperoxia after birth. Pulmonary artery Doppler measurements, micro-CT barium angiograms and media thickness of peripheral pulmonary arteries were affected after seven days of hyperoxia when compared to controls. The parenchyma was also affected both at the functional and structural level. Lung function testing showed higher tissue resistance and elastance, with a decreased lung compliance and lung capacity. Histologically hyperoxia leads to fewer and larger alveoli with thicker walls, less developed distal airways and more inflammation than normoxia. In conclusion, we show that the rabbit model develops pulmonary hypertension and developmental lung arrest after preterm lung injury, which parallel the early changes in human BPD. Thus it enables the testing of pharmaceutical agents that target the cardiovascular compartment of the lung for further translation towards the clinic., Competing Interests: The authors declare no conflict of interest. Jan Deprest is beneficiary of a fundamental clinical research grant of the Fonds Wetenschappelijk Onderzoek Vlaanderen (1801207), Jaan Toelen from the “Klinische Opleidings-en Onderzoeks-Raad” of the University Hospitals Leuven. Our experimental program is supported by the Flemish Hercules foundation (large infrastructure investments AKUL/09/033), by the KU Leuven (OT/13/115) and Julio Jimenez by the European Commission via its Erasmus Joint Doctoral program (2013-0040). This publication represents the views of the authors. The EC cannot be held responsible from any use which may be made from the information contained therein.

Objective: The objective of this study is to identify and systematically review in vivo animal studies on antenatal medical interventions to prevent bronchopulmonary dysplasia., Methods: An automated literature search was conducted using MEDLINE (Pubmed) and Embase including all studies using Medical Subject Headings (MeSH) and keywords following a step-by-step approach. All in vivo prenatal intervention studies in animal models mimicking key aspects of the pathophysiology of bronchopulmonary dysplasia were included. In view of relevance of the findings, an additional criterion was that outcomes at 48 h of life or beyond were available. The PRISMA statement concerning systemic reviews was applied and a quality checklist developed by the CAMARADES group was used., Results: In total, 518 abstracts were identified yet only eight studies were eligible for further analysis. Four studies involved administration of glucocorticoids, the other studies described therapy with epidermal growth factor, interleukin 1b, beta-naphthoflavone, or vitamin D. Outcomes were survival, pulmonary histology, lung function, and/or biochemical analysis., Conclusions: Though many in vivo experimental studies in animal models for bronchopulmonary dysplasia have been done, only few have looked into the effect of prenatal interventions and measured outcomes after at least 48 h of life. Most involve the use of antenatal glucocorticoids, although still only four.

Background: Caffeine is a commonly used drug for apnea of prematurity. It may, however, also have a beneficial effect on bronchopulmonary dysplasia (BPD), which is the most common complication of extreme preterm birth., Objectives: To study the inflammatory, structural and functional effects of caffeine in an animal model of BPD., Methods: Preterm New Zealand-Dendermonde rabbits (gestational day 28; term 31) were randomized to three groups: normoxia-placebo (N-P), hyperoxia-placebo (H-P) and hyperoxia-caffeine (H-C). Lung function was assessed on postnatal day 5, along with airway morphometry, vascular morphometry and a score observing airway inflammation., Results: Caffeine improved lung function by increasing lung volume [mean displaced volume N-P: 40.1 ± 6 ml/kg, H-P: 27.8 ± 8 ml/kg and H-C: 34.4 ± 7 ml/kg (p < 0.05); total lung capacity: N-P: 1.17 ± 0.1 ml, H-P: 0.67 ± 0.1 ml and H-C: 1.1 ± 0.1 ml (p < 0.05)], decreasing tissue damping [N-P: 2.7 ± 0.3 cm H2O/ml, H-P: 4.6 ± 0.6 cm H2O/ml and H-C: 3.2 ± 0.4 cm H2O/ml (p < 0.05)], elastance [N-P: 9.3 ± 2.4 cm H2O/ml, H-P: 19.2 ± 7.4 cm H2O/ml and H-C: 10.7 ± 2 cm H2O/ml (p < 0.05)] and compliance [N-P: 0.06 ± 0.01 cm H2O/ml, H-P: 0.054 ± 0.01 cm H2O/ml and H-C: 0.07 ± 0.013 cm H2O/ml (p < 0.05)]. Caffeine also improved histology by decreasing alveolar size [linear intercepts; N-P: 83.6 ± 1.7, H-P: 82.9 ± 1.6 and H-C: 67.3 ± 1.4 (p < 0.05)], increasing radial alveolar count (N-P: 6.6 ± 0.5, H-P: 5.7 ± 0.6 and H-C: 7.05 ± 0.5) and decreasing the acute inflammation score [N-P: 0.3 ± 0.1, H-P: 0.5 ± 0.1 and H-C: 0.4 ± 0.1 (p < 0.05)]., Conclusion: In preterm rabbits, caffeine reduces the functional, architectural and inflammatory pulmonary changes induced by hyperoxia in the lung., (© 2016 S. Karger AG, Basel.)

The objective of this study was to document early neonatal (7 days) pulmonary outcome in the rabbit model for preterm birth and hyperoxia-induced lung injury. Preterm pups were delivered at 28 days (term = 31 days; early saccular phase of lung development) by cesarean section, housed in an incubator, and gavage fed for 7 days. Pups were divided into the following groups: 1) normoxia (21% O2; normoxia group) and 2) and hyperoxia (>95% O2; hyperoxia group). Controls were pups born at term who were housed in normoxic conditions (control group). Outcome measures were survival, pulmonary function tests using the whole body plethysmograph and forced oscillation technique, and lung morphometry. There was a significant difference in survival of preterm pups whether they were exposed to normoxia (83.3%) or hyperoxia (55.9%). Hyperoxic exposure was associated with increased tissue damping and elasticity and decreased static compliance compared with normoxic controls (P < 0.01). Morphometry revealed an increased linear intercept and increased mean wall transection length, which translates to larger alveoli with septal thickening in hyperoxia compared with normoxia (P < 0.01). In conclusion, the current experimental hyperoxic conditions to which preterm pups are exposed induce the typical clinical features of bronchopulmonary dysplasia. This model will be used to study novel preventive or therapeutic interventions.