simplifying-transcutaneous-intratracheal-drug-delivery-newborn

Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and ...

KU Leuven

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.

Local pulmonary drug delivery in the preterm rabbit: feasibility and efficacy of daily intratracheal injections.

Bronchopulmonary dysplasia continues to cause important respiratory morbidity throughout life, and new therapies are needed. The common denominator of all BPD cases is preterm birth, however most preclinical research in this area focusses on the effect of hyperoxia or mechanical ventilation. In this study we investigated if and how prematurity affects lung structure and function in neonatal rabbits.

Preterm birth impairs postnatal lung development in the neonatal rabbit model.

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), or hyperoxia plus 4 h daily CPAP [fraction of inspired oxygen (Fi) 0.95, 5 cmHO]. On  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.

Intermittent CPAP limits hyperoxia-induced lung damage in a rabbit model of bronchopulmonary dysplasia.

Recent clinical trials have shown improvements in neonatal outcomes after intratracheal administration of a combination of budesonide/surfactant (ITBS) in infants at risk of bronchopulmonary dysplasia. However, the effect of ITBS on lung function and alveolar structure is not known. We aimed to determine the effect of ITBS on lung function, parenchymal structure, and inflammatory cytokine expression in a relevant preterm animal model for bronchopulmonary dysplasia. Premature neonatal rabbits were administered a single dose of ITBS on the day of delivery and exposed to 95% oxygen. Following 7 days of hyperoxia, in vivo forced oscillation and pressure-volume maneuvers were performed to examine pulmonary function. Histological and molecular analysis was performed to assess alveolar and extracellular matrix (ECM) morphology, along with gene expression of connective tissue growth factor (CTGF), IL-8, and CCL-2. ITBS attenuated the functional effect of hyperoxia-induced lung injury and limited the change to respiratory system impedance, measured using the forced oscillation technique. Treatment effects were most obvious in the small airways, with significant effects on small airway resistance and small airway reactance. In addition, ITBS mitigated the decrease in inspiratory capacity and static compliance. ITBS restricted alveolar septal thickening without altering the mean linear intercept and mitigated hyperoxia-induced remodeling of the ECM. These structural changes were associated with improved inspiratory capacity and lung compliance. Gene expression of CTGF, IL-8, and CCL-2 was significantly downregulated in the lung. Treatment with ITBS shortly after delivery attenuated the functional and structural consequences of hyperoxia-induced lung injury to  of life in the preterm rabbit.

Intratracheal budesonide/surfactant attenuates hyperoxia-induced lung injury in preterm rabbits.

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.

A semi-automated method for unbiased alveolar morphometry: Validation in a bronchopulmonary dysplasia model.

Prematurely born infants regularly develop respiratory distress syndrome and require assisted ventilation. Ventilation may injure the premature lung and increase the risk of bronchopulmonary dysplasia. Continuous positive airway pressure (CPAP), a form of noninvasive ventilation, is commonly used in modern neonatology. Limited clinical data are available on the acute and long-term effect of neonatal exposure to CPAP on the lung. Given the restricted clinical data, newborn animal models have been used to study the influence of CPAP on lung structure and function. The findings of animal studies can guide neonatal care and improve the use of CPAP.

A Systematic Review of the Influence of Continuous Positive Airway Pressure on Fetal and Newborn Animal Models: Suggestions to Improve Neonatal Respiratory Care.

Recent advances in neonatal care have improved the survival rate of those born premature. But prenatal conditions, premature birth and clinical interventions can lead to transient and permanent problems in these fragile patients. Premature birth (<36 gestational weeks) occurs during critical renal development and maturation. Some consequences have been observed but the exact pathophysiology is still not entirely known. This experimental animal study aims to investigate the effect of premature birth on postnatal nephrogenesis in premature neonatal rabbits compared to term rabbits of the same corrected age. We analyzed renal morphology, glomerular maturity and functional parameters (proteinuria and protein/creatinine ratio) in three cohorts of rabbit pups: preterm (G28), preterm at day 7 of life (G28+7) and term at day 4 of life (G31+4). We found no significant differences in kidney volume and weight, and relative kidney volume between the cohorts. Nephrogenic zone width increased significantly over time when comparing G31 + 4 to G28. The renal corpuscle surface area, in the inner cortex and outer cortex, tended to decrease significantly after birth in both preterm and term groups. With regard to glomerular maturity, we found that the kidneys in the preterm cohorts were still in an immature state (presence of vesicles and capillary loop stage). Importantly, significant differences in proteinuria and protein/creatinine ratio were found. G28 + 7 showed increased proteinuria (p = 0.019) and an increased protein/creatinine ratio (p = 0.023) in comparison to G31 +4. In conclusion, these results suggest that the preterm rabbit kidney tends to linger in the immature glomerular stages and shows signs of a reduced renal functionality compared to the kidney born at term, which could in time lead to short- and long-term health consequences.

Glomerular developmental delay and proteinuria in the preterm neonatal rabbit.

Various lung diseases, including pulmonary hypertension, chronic obstructive pulmonary disease or bronchopulmonary dysplasia, are associated with structural and architectural alterations of the pulmonary vasculature. The light microscopic (LM) analysis of the blood vessels is limited by the fact that it is impossible to identify which generation of the arterial tree an arterial profile within a LM microscopic section belongs to. Therefore, we established a workflow that allows for the generation-specific quantitative (stereological) analysis of pulmonary blood vessels. A whole left rabbit lung was fixed by vascular perfusion, embedded in glycol methacrylate and imaged by micro-computed tomography (µCT). The lung was then exhaustively sectioned and 20 consecutive sections were collected every 100 µm to obtain a systematic uniform random sample of the whole lung. The digital processing involved segmentation of the arterial tree, generation analysis, registration of LM sections with the µCT data as well as registration of the segmentation and the LM images. The present study demonstrates that it is feasible to identify arterial profiles according to their generation based on a generation-specific color code. Stereological analysis for the first three arterial generations of the monopodial branching of the vasculature included volume fraction, total volume, lumen-to-wall ratio and wall thickness for each arterial generation. In conclusion, the correlative image analysis of µCT and LM-based datasets is an innovative method to assess the pulmonary vasculature quantitatively.

Article	Year
Local pulmonary drug delivery in the preterm rabbit: feasibility and efficacy of daily intratracheal injections. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 30675804	2019
Preterm birth impairs postnatal lung development in the neonatal rabbit model. Respiratory research\| PubMed ID: 32085773	2020
Intermittent CPAP limits hyperoxia-induced lung damage in a rabbit model of bronchopulmonary dysplasia. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 32186390	2020
Intratracheal budesonide/surfactant attenuates hyperoxia-induced lung injury in preterm rabbits. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 32903026	2020
A semi-automated method for unbiased alveolar morphometry: Validation in a bronchopulmonary dysplasia model. PloS one\| PubMed ID: 32966330	2020
A Systematic Review of the Influence of Continuous Positive Airway Pressure on Fetal and Newborn Animal Models: Suggestions to Improve Neonatal Respiratory Care. Neonatology\| PubMed ID: 33091899	2020
Glomerular developmental delay and proteinuria in the preterm neonatal rabbit. PloS one\| PubMed ID: 33166318	2020
Combination of µCT and light microscopy for generation-specific stereological analysis of pulmonary arterial branches: a proof-of-concept study. Histochemistry and cell biology\| PubMed ID: 33263790	2021

Andre Gie

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