Characterization of a Preterm Rabbit Model Exposed to Hyperoxia for Preclinical Studies of Bronchopulmonary Dysplasia (BPD)

Background: Bronchopulmonary dysplasia (BPD) is the most common long-term respiratory complication of prematurity. Its pathogenesis is multifactorial, with several pre- and post-natal insults contributing to the arrest of normal lung development. Due to the limited availability of lung samples from patients with BPD, animal models still play an essential role for investigating the pathogenic mechanisms and evaluating potential pharmacological interventions. Among the existing animal models, the preterm rabbit represents a good compromise between small (mice and rats) and large (baboons and lambs) animal species. Hyperoxia exposure remains the most common postnatal insult, used in combination with preterm birth, to reproduce key aspects of BPD pathophysiology. At Chiesi’s Research Centre, the preterm rabbit model exposed to hyperoxia (95% O2) for seven days has been set-up and validated. This thesis aimed to assess the effects of prematurity and hyperoxia exposure on postnatal lung development through functional, histological and biomolecular analyses, providing a comprehensive characterization of the hyperoxia-exposed preterm rabbit model of BPD. Materials and Methods: Preterm rabbit pups were delivered by cesarean section (C-section) on gestational day 28 and randomized to hyperoxia (95% O2) and normoxia (21% O2) for three and seven days. Pups born at term on gestational day 31 and those left with their mothers at room air for four days after natural delivery were included as age-matched controls for physiological postnatal development. At the end of each experimental period, lung function was measured using the flexiVentTM system. Histological analyses were performed through conventional morphometric techniques and an innovative Artificial Intelligence (AI)-based software (Visiopharm®). Immunohistochemistry (IHC) for Surfactant Protein-C (SP-C) and α-Smooth Muscle Actin (α-SMA) was performed by using an automated research stainer. Two applications were developed: one for the quantification of SP-C positive type II alveolar epithelial (AT II) cells and another for the measurement of the thickness of α-SMA positive tunica media in small pulmonary blood vessels. Ultrastructural alterations in the alveolar epithelium and in the pulmonary vascular wall were analyzed by Transmission Electron Microscope (TEM). Functional and histological data were then integrated with biomolecular analysis by quantitative Real TimePCR (qRT-PCR) to assess the mRNA expression level of genes involved in vascular development and inflammatory response. Results: Preterm pups exposed to hyperoxia exhibited significantly impaired lung function, as evidenced by decreased inspiratory capacity and static compliance and increased tissue damping and tissue elastance. Morphologically, hyperoxic lungs showed histopathological features characteristic of BPD, including enlarged airspaces, thickened alveolar septa, infiltration of inflammatory cells, accumulation of proteinaceous debris and disrupted elastic fiber architecture. Conventional morphometric analyses demonstrated a significant increase in Mean Linear Intercept (MLI), Acute Lung Injury (ALI) score, together with a decrease in Secondary Crests (SCs) count and Radial Alveolar Count (RAC) after postnatal exposure to hyperoxia. AI-based quantitative analysis confirmed the reduction in SCs count and showed a significant increase in Septal Thickness (ST). Moreover, in preterm pups exposed to hyperoxia, a significantly decreased number of AT II cells and increased thickness of the tunica media of small pulmonary blood vessels were observed. These results were subsequently confirmed by ultrastructural analysis, which showed morphological alterations in the vascular wall, as well as a reduction in the number of mature AT II cells. Postnatal exposure to hyperoxia also induced an imbalance of angiogenesisregulating genes favoring anti-angiogenic factors and an upregulation of genes coding for proinflammatory cytokines. Conclusions: The preterm rabbit model exposed to hyperoxia successfully mimics functional, structural and biomolecular features of the human disease, highlighting its translational relevance for preclinical studies of BPD.