Preterm rabbit-derived Precision Cut Lung Slices as alternative model of bronchopulmonary dysplasia in preclinical study: a morphological fine-tuning approach

Bronchopulmonary dysplasia (BPD) is the prevalent sequela of preterm birth, associated with appreciable morbidity and mortality in neonatal intensive care (Gisondo and Donn, 2020, Nardiello et al., 2017). BPD has changed its histopathological characteristics over the years. It initially occurred mainly in moderately preterm infants undergoing aggressive mechanical ventilation and exposure to high oxygen concentrations, which could cause a combination of inflammation, oxygen toxicity, barotrauma, volutrauma, and infections leading to lung fibrosis and scarring ("old "BPD) (Day and Ryan, 2017). Recent advances in neonatal care have given greater chances of survival to extremely premature infants benefiting from antenatal steroids, surfactants and minimally invasive supportive measures, but have also increased the number of individuals showing a significant arrest of postnatal growth and maturation of the still developing lung. The lungs of patients affected by this "new" BPD (Day and Ryan, 2017) have fewer alveoli, wider and with thicker walls, therefore reduced total gas exchange surface and hindered gas diffusion compared to normal lungs. In addition, dysmorphic pulmonary vessels develop, which complicate blood flow through the lung, causing hypertension (Niedermaier and Hilgendorff, 2015). In case of survival, the abnormalities of the angiogenic process persist into adulthood and pulmonary fibrosis and thickening of the submucosal layers are consistent with chronic obstructive pulmonary disease, making BPD also a chronic lung disease (Gisondo and Donn, 2020, Nardiello et al., 2017).

Understanding how the alveoli and capillary network develop and how these mechanisms are disrupted in BPD is crucial for developing efficient therapies. The use of an effective and appropriate animal model of the disease in preterm infants is the key to the research (Salaets et al., 2017).

Relatively short gestation and postnatal lung maturation times, readiness and ease of maintenance made mice and rats the preferred species for BPD studies compared to larger vertebrates (e.g. lambs or non-human primates), about which regulatory agencies for experimental studies raise even greater concerns. However, some doubts have been raised about the translational limitations of rodent models to human BPD. In fact, premature children prone to developing BPD are born during the canalicular-saccular phase of lung development, when branched morphogenesis and alveolar differentiation have yet to be completed. In newborn rodents the lungs are in the saccular phase of development, but are functionally ready for breathing. Therefore, they lack prematurity and show no signs of respiratory distress. Even the small size of mice and rats at birth, while allowing the use of smaller quantities of pharmacological agents than those required for larger animals, make these animals particularly difficult to intubate. This is important, as mechanical ventilation is a very clinically relevant harmful stimulus for BPD (D’Angio and Ryan, 2014). In this scenario, a good compromise between translational and practical considerations may be represented by medium-size animals, such as rabbits, which combine prematurity, the possibility of perinatal manipulation and relatively low costs. Lungs of preterm rabbit fetuses delivered by caesarean section on day 28 of gestation exhibit a phenotype comparable to respiratory distress syndrome (RDS), even in the absence of respiratory support and prenatal injury (Salaets et al., 2020, Salaets et al., 2017).

In preclinical research, great attention is then given to replacing animal models with alternative assays, reducing the number of animals used in experiments and, where possible, optimizing the experimental procedures, in accordance with the 3 R (Replacement, Reduction and Refinement) principle. The use of in vitro assays based on ex vivo culture of Precision Cut Lung Slices (PCLS) obtained from lungs previously inflated with agarose, in an attempt to preserve distal airway structure, goes precisely in this direction. It represents a good compromise between the well controllable and flexible in vitro models and the more physiologically relevant in vivo ones. Unlike cell cultures, PCLS “contain all different types of organ cells in vivo, in their normal spatial relationship and with the same potential for normal cell-cell and cell-matrix interaction” (Bach et al., 1996). Therefore, they offer a good representation of the complexity of the intact organ, allowing, at the same time, the examination of the local response in the lung without the complicated influences of the immune or systemic physiological response (i.e. in the absence of recruited cells, such as monocytes, lymphocytes and neutrophils) (Liberati et al., 2010).

PCLS have rarely been used for the analysis of lung development and related diseases. Most often they have been used in studies concerning airway reactivity and injury and have been obtained from the lungs of adult individuals, therefore fully developed and with low probability of showing alveolar development (Sanderson, 2011). PCLS derived from premature rabbits have never been used in preclinical studies of BPD. A morphological analysis of their parenchymal structures during the days in culture and of the effect of different oxygen conditions, which is the goal of this study, has not yet been fine-tuned (Liu et al., 2019).

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