RESOURCE ARTICLE

Standardisation of oxygen exposure in the development of mousemodels for bronchopulmonary dysplasiaClaudio Nardiello1,2, IvanaMizıkova1,2, DiogoM. Silva1,2, Jordi Ruiz-Camp1,2, Konstantin Mayer2, Istvan Vadasz2,Susanne Herold2, Werner Seeger1,2 and Rory E. Morty1,2,*

ABSTRACTProgress in developing new therapies for bronchopulmonary dysplasia(BPD) is sometimes complicated by the lack of a standardised animalmodel. Our objective was to develop a robust hyperoxia-based mousemodel of BPD that recapitulated the pathological perturbations to lungstructure noted in infants with BPD. Newborn mouse pups wereexposed to a varying fraction of oxygen in the inspired air (FiO2) and avarying window of hyperoxia exposure, after which lung structure wasassessed by design-based stereology with systemic uniform randomsampling. The efficacy of a candidate therapeutic intervention usingparenteral nutrition was evaluated to demonstrate the utility of thestandardised BPDmodel for drug discovery. AnFiO2 of 0.85 for the first14 days of life decreased total alveoli number and concomitantlyincreased alveolar septal wall thickness, which are two keyhistopathological characteristics of BPD. A reduction in FiO2 to 0.60or 0.40 also caused a decrease in the total alveoli number, but theseptal wall thickness was not impacted. Neither a decreasing oxygengradient (from FiO2 0.85 to 0.21 over the first 14 days of life) nor anoscillation in FiO2 (between 0.85 and 0.40 on a 24 h:24 h cycle) had anappreciable impact on lung development. The risk ofmissing beneficialeffects of therapeutic interventions at FiO2 0.85, using parenteralnutrition as an intervention in the model, was also noted, highlightingthe utility of lower FiO2 in selected studies, and underscoring the needto tailor the model employed to the experimental intervention. Thus,a state-of-the-art BPD animal model that recapitulates the twohistopathological hallmark perturbations to lung architectureassociated with BPD is described. The model presented here, whereinjurious stimuli have been systematically evaluated, provides a mostpromising approach for the development of new strategies to drivepostnatal lung maturation in affected infants.

KEY WORDS: BPD, Hyperoxia, Alveolarisation, Structure, Animalmodel

INTRODUCTIONPrecise modelling of human disease using animal models is a majorchallenge in translating bench science to the bedside, as (1) animalmodels must accurately recapitulate disease processes to facilitate

the identification of pathogenic pathways, and (2) animal modelsrepresent the limiting step in assessing which therapeuticinterventions hold promise for subsequent study. This isparticularly evident in animal models of human diseases that arecharacterised by perturbations to the architecture of an organ.Modelling disease pathogenesis in experimental animals isproblematic from multiple perspectives. Amongst these, theinjurious insult employed in the experimental model might notrecapitulate key elements of disease, thereby limiting the ability toevaluate the efficacy of candidate therapeutic agents. Furthermore,the precision of the readout that is employed might be inadequate todetect small changes in anatomical structures that are targetedby both the injurious insult and candidate therapeutic intervention.A further confounding variable is the use of experimental animals inmedical research, as emphasis must be placed on ‘reduction,refinement and replacement’ (the 3R concept) (Curzer et al., 2016),where the number of experimental animals employed and the levelof stress to which the animal is subjected must be maintained at theminimum level possible, while still retaining the translationalviability of the animal model.

Modelling bronchopulmonary dysplasia (BPD) in experimentalanimals is a textbook illustration of these concerns. BPD is the mostcommon complication of preterm birth and represents significantmorbidity and mortality in the neonatal intensive care unit (Jobe,2011; Jobe and Tibboel, 2014). BPD is caused by a combination ofthe toxic effects of oxygen supplementation used to managerespiratory failure in preterm infants, baro- and volu-trauma frommechanical ventilation (Greenough et al., 2008), as well as otherdisease-modifying variables such as infection and inflammation(Balany and Bhandari, 2015). BPD results in long-termcomplications in respiratory function that persist into adulthood(Hilgendorff and O’Reilly, 2015). The pathology of BPD haschanged over time; where ‘old’ BPD, which is particularlycharacterised by fibrosis, thickened septa and some alveolarsimplification, results from aggressive mechanical ventilation withhigh oxygen levels. In contrast, ‘new’ BPD, which is the prevalentform today, is largely characterised by alveolar simplification,where preterm infants are less aggressively ventilated, with loweroxygen levels (Jobe, 2011). As a result of improvements in themedical management of BPD, the incidence of BPD is increasingbecause preterm infants delivered earlier have increasinglyimproved survival (Stoll et al., 2015). This underscores a pressingneed to develop new medical management strategies. However,these efforts might be hampered by the lack of appropriate animalmodels of BPD. In affected individuals, two key elements of thelung structure are impacted: the oxygen supplementation arrestslung development, which causes fewer alveoli of a larger size to begenerated. Concomitantly, the thickness of the delicate barrierbetween the alveolar airspaces in the lung and the capillary networkof the lung is thickened, which compromises gas exchange, and isReceived 9 July 2016; Accepted 24 November 2016

1Department of Lung Development and Remodelling, Max Planck Institute for Heartand Lung Research, 61231 Bad Nauheim, Germany. 2Department of InternalMedicine (Pulmonology), University of Giessen andMarburg Lung Center (UGMLC),member of the German Center for Lung Research (DZL), 35392 Giessen, Germany.

*Author for correspondence (rory.morty@mpi-bn.mpg.de)

R.E.M., 0000-0003-0833-9749

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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evident by thickened alveolar septal walls. In order to properlymodel BPD, the injurious intervention (oxygen supplementation)must recapitulate both structural elements of the pathology, adecreased number of alveoli and an increased alveolar septal wallthickness.To this end, a large number of methodologies have been reported

that rely on the exposure of newborn mouse pups to an increasedfraction of oxygen in the inspired air (FiO2), often reported as apercentage of oxygen in the inspired air, which ranges between 40%O2 (FiO2 0.4) and 100% O2 (FiO2 1.0) (Silva et al., 2015). Between1 January 2013 and 30 June 2015, 41 different oxygen exposureprotocols had been reported in BPD animal models (Silva et al.,2015), sometimes with diametrically opposite findings concerningthe effects of the same intervention in different animal models ofBPD (for example, compare the studies of Britt et al., 2013 andMasood et al., 2014). This underscores the impact of the oxygenexposure regimen selected on data analysis and study conclusions.Of course, it must be acknowledged that diversity in animal modelsis also a strength of the BPD field, where BPD has a multifactorialaetiology that cannot be fully modelled in a single, standardisedanimal model.The analysis of lung structure in material from animal models of

BPD is also problematic (Silva et al., 2015). Currently, a mixture ofapproaches is employed, often based on the analysis of paraffin-embedded lung tissue with classical determinants of mean linearintercept (MLI) and radial alveolar count (RAC) as surrogates of thesize of the alveoli (Silva et al., 2015). These determinations aremade by direct measurement of the distance between the adjacentwalls of an alveolus. Similarly, the thickness of the alveolar wall isdirectly assessed using a slide-rule and visual inspection, which isnot unbiased. Furthermore, the highly elastic structure of the lungresults in substantial distortion of the lung structure during thedehydration and rehydration of the lung during paraffin embedding(Schneider and Ochs, 2014). These are all important concerns in theanalysis of the lung architecture, although these approaches havebeen successfully used to identify potentially important pathogenicpathways and for the pre-clinical evaluation of candidate therapeuticinterventions (Liao et al., 2015; Olave et al., 2016; Tibboel et al.,2013).To address the concerns outlined above, important advances have

been made replacing paraffin embedding with plastic embedding,along with treatment of lung tissue with arsenic, osmium, anduranium, which results in appreciable preservation of the lungstructure (Schneider and Ochs, 2014). Furthermore, a design-basedstereological approach to the analysis of organ structure has beendeveloped, and continues to be refined (Schneider and Ochs, 2013;Tschanz et al., 2014). This approach represents a substantialadvance over the MLI and RAC methods when applied to the lung,as the technique is both unbiased (thus not subjective), and has avery high resolution (Mühlfeld and Ochs, 2013; Ochs andMühlfeld, 2013). Design-based stereology has recently beenapplied to the analysis of the architecture of developing lungs ofnewborn mouse pups by the authors, for the first time allowing theassessment of the total number of alveoli in the lung, and theassessment of changes <1 µm in magnitude during perturbations tonewborn mouse lung development (Madurga et al., 2015, 2014;Mižíková et al., 2015).These recent developments in tissue embedding and structural

analysis represent an important advance in our ability to study lungdevelopment. Here, we employed state-of-the-art tissue embeddingmethodology, together with state-of-the-art design-basedstereology, to identify the magnitude of oxygen toxicity and the

window of oxygen exposure that is required to model BPD asperfectly as possible in mice. The primary threshold parametersselected were a decrease in total alveoli number in the lung, andan increase in the alveolar septal wall thickness, which are thetwo anatomical hallmarks of BPD. We then documented theextraordinary impact of using different oxygen levels inthe experimental model on the observed efficacy of a candidateintervention, thus highlighting the crucial importance of selectingthe correct model to evaluate experimental therapeutics.

RESULTSTo identify the optimal injurious levels of oxygen, as well as theoptimal window of exposure to injury, a series of oxygen exposureprotocols were employed, which varied the FiO2 over the first14 days of life (Fig. 1A) in a variety of windows of exposure(Fig. 1B).

Identification of the optimal level of oxygen required tomodel BPD in miceFourteen days after birth [postnatal day (P)14], the bulk of postnatallung alveolarisation has been completed (Burri, 2006). Thus,maintaining newborn mouse pups (n=5 per experimental group)under room-air (21% O2) conditions, starting on the day of birth, upto and including P14, served as the control protocol and generatedmouse lungs that exhibited an anatomically normal alveolarstructure (Fig. 2A,G). Increasing the concentration of oxygen inthe inspired air to 40% O2 over the first 14 days of life generated aless organised lung parenchymal architecture (Fig. 2B,H versusFig. 2A,G) and decreased the total number of alveoli in the lung by39% (Fig. 2M) in comparison with the 21% O2 (control) group(complete data set in Table 1). This was accompanied by aconcomitant 25% decrease in the alveolar density (Fig. 2N) and a20% decrease in the gas exchange surface area (Fig. 2O), whereasno impact on alveolar septal wall thickness was noted (Fig. 2Q).Increasing the concentration of oxygen in the inspired air to 60% O2

over the first 14 days of life did not further impact the visualappearance of the lung structure (Fig. 2C,I), which exhibited acomparable number of alveoli, alveolar density, gas exchangesurface area, and alveolar septal wall thickness to the 40% O2 group(Fig. 2M-O,Q,R); however, the meanMLI was increased in the 60%O2 group (Fig. 2P). In contrast, newborn mouse pups exposed to85% O2 for the first 14 days of life exhibited further reductions of69% in number of alveoli (Fig. 2M), 69% in alveolar density(Fig. 2N) and 37% in gas exchange surface area (Fig. 2O) comparedwith the 21% O2 control group. An increase in alveolar septal wallthickness of 25% in comparison with 21% O2 controls was alsonoted (Fig. 2Q). Thus, 85% O2 was the only oxygen concentrationthat impacted both total number of alveoli as well as alveolar septalwall thickness. For this reason, we performed two additionalstudies: the exposure of newborn mouse pups to a gradient ofdecreasing oxygen concentration from 85%O2 on the day of birth to21% O2 on P14 (amounting to a step-wise decrease of 5% O2 perday), as well as an oscillation between 85% O2 and 40% O2 on a24 h:24 h oscillating cycle (Fig. 1A).

The exposure of newborn mouse pups to a decreasing gradient ofoxygen between 85% O2 and 21% O2 over the first 14 days of lifehad no impact on any parameter of lung architecture (Fig. 2E,K,M-R).In contrast, exposure of newborn mouse pups to an oscillation of85% O2 and 40% O2 over the first 14 days of life generated lungsthat were impacted on visual inspection, albeit lightly (Fig. 2F,L);and which exhibited a comparatively moderate reduction of 25% inthe total number of alveoli in the lung (Fig. 2M) and 20% reduction

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in alveolar density (Fig. 2N), which was insufficient to significantlyimpact the gas exchange surface area, which remained unchanged(Fig. 2O). Similarly, alveolar septal wall thickness (Fig. 2Q) wasunaffected. Changes in the MLI parameter largely paralleled thosenoted in other lung structure parameters (Fig. 2P). It is important tonote that the MLI reported here represents a stereologicallydetermined mean MLI over the whole lung, and not the potentiallybiased MLI determined by visual inspection of selected lungsections, which is widely reported in the literature. No change in lungvolume was noted at P14 comparing all experimental groups(Fig. 2R), and none of the parameters assessed exhibited anyclustering on the basis of the sex of the animals (Fig. 2M-R).Together, these data demonstrate that exposure of newborn

mouse pups to as little as 40% O2 over the first 14 days of life couldrecapitulate the changes seen in lung alveolar number in individualswith BPD; however, only exposure to 85% O2 could alsorecapitulate the changes noted in lung alveolar septal wallthickness in individuals with BPD.

Identification of the optimal window of oxygen exposurerequired to model BPD in miceGiven that only exposure of newborn mouse pups to 85% O2 overthe first 14 days of life could recapitulate the two hallmarkperturbations to lung architecture seen in BPD, we then set out toassess the critical (and minimal) window of exposure to 85% O2,

comparing five different windows of exposure (Fig. 1B) over thefirst 14 days of life (n=5 per experimental group). Exposure ofnewborn mouse pups to 85% O2 for the first 24 h of life only(followed by 13 days of 21% O2) had no impact on lung structure,either by visual inspection (Fig. 3B,H), or a design-based stereologyanalysis of the number of alveoli in the lung, the alveolar density,the gas exchange surface area, the MLI, or the alveolar septal wallthickness (Fig. 3M-R; complete data sets in Table 2). Increasing thewindow of exposure to include the first 72 h of life, from P1 up toand including P3, followed by 11 days of 21% O2 (Fig. 3C,I)generated a moderate (14%) decrease in alveolar density incomparison with the 21% O2 group (Fig. 3N), without impactingany other parameter. When an alternative 72 h window of exposure,where mouse pups were exposed to 85% O2 over a time-framestarting with and including P4, up to and including P7 was selected,a 32% decrease in the number of alveoli (Fig. 3M) and a 39%decrease in the alveolar density (Fig. 3N) in comparison with the21% O2 group was noted. Although these changes were notsufficient to impact the gas exchange surface area (Fig. 3O), anincrease in alveolar septal wall thickness of 19%, in comparisonwith 21% O2 was also noted (Fig. 3Q).

Exposure of newborn mouse pups either to (1) 85% O2 from P1to P7, followed by seven days of 21% O2 (Fig. 3E,K) or (2) to 85%O2 from P1 to P14 (Fig. 3F,L) generated the most dramatic impacton the lung architecture. A decrease in number of alveoli, alveolar

Fig. 1. Schematic illustration of the different oxygen exposure protocols employed. (A) Determination of the effects of the level of oxygen employed as aninjurious stimulus. Exposure protocols include the continuous exposure of newborn mouse pups from postnatal day (P)1 (the day of birth) to the end of P14, toambient room air (21% O2), 40% O2, 60% O2, and 85% O2. Alternatively, newborn mouse pups were exposed to a decreasing gradient of O2 concentration from85%O2 at P1 to 21%O2 at P14; or to an oscillation between 85%O2 and 40%O2 in an oscillation cycle of 24 h: 24 h, starting with 85%O2 on P1, and ending with40% O2 on P14. (B) Determination of the effects of the time frame (window) of exposure to injurious oxygen levels. The exposure protocols included thecontinuous exposure of newborn mouse pups from P1 to the end of P14 to ambient room air (21%O2); to 85%O2 for the first 24 h of life, followed by 21%O2 fromthe start of P2 to the end of P14; to 85%O2 from P1 to the end of P3 followed by 21%O2 from the start of P4 to the end of P14; to 21%O2 from P1 to the end of P3,followed by 85% O2 from the start of P4 to the end of P7, followed by 21% O2 to the end of P14; to 85% O2 from P1 to the end of P7, followed by 21% O2 from thestart of P8 to the end of P14; or to 85% O2 from P1 to the end of P14.

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density, and alveolar surface area, as well as an increase in MLIand alveolar septal wall thickness was noted in both groups(Fig. 3M-R), suggesting that these two protocols best model BPDin mice.

Effect of oxygen exposureon theperformanceof acandidatetherapeutic interventionCottonseed oil was employed as an intervention for parenteralnutrition, and exhibited no impact on the structural development of

Fig. 2. Optimising oxygen exposure levels to model bronchopulmonary dysplasia in newborn mice. Histological images were obtained from the lungs ofmice subjected to the six oxygen exposure protocols illustrated in Fig. 1A. (A-F) Low-magnification images from the lungs, which were embedded in glycolmethylacrylate plastic after fixation with buffered paraformaldehyde/glutaraldehyde and treatment with sodium cacodylate, osmium tetroxide, and uranyl acetate,then stained with Richardson’s stain. (G-L) Higher-magnification images derived from part (demarcated by the red box) of the corresponding images to the left, tohighlight changes in alveolar septal wall thickness. Each image is representative of lung sections obtained from four other mouse pups within each experimentalgroup (n=5, per group). Scale bars: 200 µm. (M-Q) Design-based stereology was employed to assess (M) total number of alveoli in the lung, (N) alveolar density,(O) gas exchange surface area, (P) mean linear intercept, and (Q) alveolar septal wall thickness. (R) The lung volume was estimated by the Cavalieri method. Inpanels M-R, • denotes male animals, ▴ denotes female animals. Data represented as mean±s.d. Data comparisons weremade by one-way ANOVAwith Tukey’spost hoc test. Significant P-values are indicated in the graphs; n.s., not significant (P≥0.05).

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newborn mouse lungs over the period P1 to P14 in mouse pups (n=5per experimental group) maintained under 21% O2 compared withthe lungs of control sham-treated mice maintained under 21% O2

(Fig. 4C,D versus Fig. 4A,B; Fig. 4M-R; complete data set inTable 3). Cottonseed oil tended to increase body mass (by up to 20%)and had no impact on survival (data not shown). When cottonseed oilwas applied to newborn mouse pups exposed to 60% O2 over theperiod P1-P14, a pronounced recovery in the number of alveoli (anincrease of 78%) was noted compared with sham-treated pups in thesame oxygen-exposure protocol (Fig. 4G,H versus Fig. 4E,F;Fig. 4M). Similarly, cottonseed oil application increased the alveolardensity by 31% (Fig. 4N) and increased the gas exchange surface areaby 55% (Fig. 4O) in the 60% O2 group. These changes wereaccompanied by an increase in lung volume of 33% (Fig. 4R). Asalveolar septal wall thickness was not impacted at 60% O2 (Fig. 2Q)no impact of cottonseed oil administration on alveolar septal wallthickness was expected, nor was anyeffect noted (Fig. 4Q). These datahighlight the therapeutic benefit of cottonseed oil supplementation inthis experimental animal model of BPD. However, when the moresevere hyperoxia exposure model was employed with continuousexposure of newborn mouse pups to 85% O2 from P1 to P14, thebeneficial impact of cottonseed oil administration on alveolar numberand alveolar density was lost (Fig. 4K,L versus Fig. 4I,J; Fig. 4M,N).However, the impact of cottonseed oil on mean septal wall thicknesswas still noted (Fig. 4Q). Taken together, these data highlight a keyconcern of the 85%O2 exposure approach, where the extreme severityof the injurious stimulus might result in promising candidatetherapeutic interventions being missed or discounted.

DISCUSSIONGiven the increasing clinical burden of BPD (Jobe, 2011; Stollet al., 2015), there is a pressing need for new therapeutic optionsfor this syndrome. Progress in this regard has been impeded by alack of suitable animal models, which represent the first step in thetranslational pipeline. Although nonhuman primates are a betterlaboratory model for these studies than are rodents (Herring et al.,2014), ethical considerations, and monetary and time costssupport the use of rodents as first-line models for drugdiscovery and studies on disease pathogenesis. Thus, mice havefound widespread application in BPD models (Berger andBhandari, 2014), particularly because of the availability oftransgenic mouse lines. Most mouse BPD models are based onexposure of newborn pups to elevated oxygen levels, as oxygentoxicity to the developing lung is the medical basis of BPD inpreterm infants (Jobe, 2011; Stoll et al., 2015), and oxygentoxicity is known to disrupt elements of alveolar developmentincluding the maturation of the extracellular matrix (Mižíková andMorty, 2015), microRNA dynamics (Nardiello and Morty, 2016),as well as stem cell plasticity (Domm et al., 2015; Yee et al.,2016). However, there is a pronounced lack of standardisation ofBPD mouse models, with over 41 different oxygen-exposureprotocols reported in a 30-month period alone (Silva et al., 2015).Although initially very high (90-100%) oxygen concentrationswere employed in the past as an injurious stimulus in BPDmodels, and indeed, still are by many groups; there has been agradual shift in the degree of oxygen toxicity employed, withincreased instances of oxygen concentrations below 80% O2

becoming increasingly evident in recent published studies (Bouchet al., 2016, 2015; Ehrhardt et al., 2016; Maduekwe et al., 2015;Reyburn et al., 2016; Sozo et al., 2015; Wang et al., 2014; Yeeet al., 2016). In some instances, the lack of model standardisationmight have resulted in opposite findings when evaluating theTa

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same candidate therapeutic intervention (Silva et al., 2015). Theseconcerns might suggest the benefits of a standardised modelof BPD.

Recent developments in the stereological analysis of tissue andorgan structure have provided a tool that can be used to studypathological changes to the architecture of a target organ – even of a

Fig. 3. Optimising the oxygen exposure window to model bronchopulmonary dysplasia in newborn mice. Histological images were obtained from thelungs ofmice subjected to the six oxygen exposure protocols illustrated in Fig. 1B. (A-F) Low-magnification images from the lungs, which were embedded in glycolmethylacrylate plastic after fixation with buffered paraformaldehyde/glutaraldehyde and treatment with sodium cacodylate, osmium tetroxide, and uranyl acetate,then stained with Richardson’s stain. (G-L) Higher-magnification images derived from part (demarcated by the red box) of the corresponding images to the left, tohighlight changes in alveolar septal wall thickness. Each image is representative of lung sections obtained from four other mouse pups within each experimentalgroup (n=5, per group). Scale bars: 200 µm. (M-Q) Design-based stereology was employed to assess (M) total number of alveoli in the lung, (N) alveolar density,(O) gas exchange surface area, (P) mean linear intercept, and (Q) alveolar septal wall thickness. (R) The lung volume was estimated by the Cavalieri method. Inpanels M-R, • denotes male animals, ▴ denotes female animals. Data represented as mean±s.d. Data comparisons weremade by one-way ANOVAwith Tukey’spost hoc test. Significant P-values are indicated in the graphs.

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very small magnitude – with extraordinarily high precision.Similarly, these design-based stereology approaches can also beused to evaluate the efficacy of candidate therapeutic interventionsthat can correct pathological perturbations to organ structure. To thisend, specific guidelines exist for the use of design-based stereologyto study lung structure (Hsia et al., 2010), and the design-basedstereology approach has been refined by the authors for the study ofnormal and aberrant lung development in the newborn mouse(Madurga et al., 2015, 2014; Mižíková et al., 2015).

Here, we report the use of design-based stereology tosystematically explore the impact of oxygen on postnatal mouselung development, considering both the level of oxygen in theinspired air, and the window of oxygen exposure. As in all organs,the structure of the lung is intimately related to lung function (Hsiaet al., 2016), and lung structure in infants with BPD exhibit twopathological hallmarks: disturbance to the gas exchange structure(cumulatively reflected by a change in total number of alveoli,alveolar density and gas exchange surface area), as well asthickening of the alveolar septal walls. Both lung structuralelements of BPD are recapitulated in the continuous exposure to85% O2 for the first 14 days of life.

We have selected P14 as the end-point for our studies as P14 is thetime-point at which the bulk of secondary septation is largelycompleted, and as such, even subtle perturbations to secondaryseptation will be suitably amplified and (relatively) easy to detect atP14. Given that secondary septation is broadly thought to extend atleast to P10, P14 seemed to be a suitable time point. Furthermore,reference to Silva et al. (2015) revealed that P14 is the mostcommonly used end point for hyperoxia-based studies. As such, thisis the best time point to terminate the studies presented here, tofacilitate comparisons with the work of others. Our data haverevealed some interesting effects of oxygen exposure protocols onthe postnatal development of the mouse lung. Continuous exposureto as little as 40% O2 over the first 14 days of postnatal life didimpact postnatal lung development, with continuous exposure to40% O2, 60% O2, and 85% O2 having an impact on the number ofalveoli (Fig. 2M), alveolar density (Fig. 2N), and gas exchangesurface area (Fig. 2O), whereas only continuous exposure to 85%O2 impacts mean septal wall thickness (Fig. 2Q). In general, theimpact of 40%O2 and 60%O2were largely comparable consideringthe effect of oxygen exposure on number of alveoli and mean septalwall thickness. Some unexpected observations were also made.Notably, a decreasing O2 gradient from 85% O2 at P1 to 21% O2 atP14 was without any impact on number of alveoli (Fig. 2M) ormean septal wall thickness (Fig. 2Q), although the cumulativeoxygen exposure exceeded that of the continuous 40% O2 groupwhere both number of alveoli and mean septal wall thickness wereimpacted. It is speculated that after birth the immediate and dramatic(85% O2) oxygen toxicity recruited anti-oxidant lung protectivepathways, which were progressively titrated down over time,commensurate with progressively decreasing O2 levels, thusmaintaining the lung protective strategies at levels that wereprotective, but never damaging. Along similar lines, oscillating ona 24 h:24 h cycle between 85% O2 and 40% O2 also represented acumulative oxygen exposure that was greater than the continuous40% O2 protocol; however, the degree of damage to the lung wascomparable, perhaps also indicating the recruitment and de-recruitment of lung protective strategies by sudden exposure todramatic changes in inspired O2 levels.

The data presented here suggest that exposure of newborn mousepups either to (1) 85% O2 from P1 to P7, followed by seven days of21% O2 (Fig. 3E,K) or (2) to 85% O2 from P1 to P14 (Fig. 3F,L)Ta

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21%

85%

P1-P14

P1

P1-P4

P4-P7

P1-P7

P1-P14

Param

eter

Mea

n±s.d.

Mea

n±s.d.

P-value

versus

21%

O2

Mea

n±s.d.

P-value

versus

21%

O2

Mea

n±s.d.

P-value

versus

21%

O2

Mea

n±s.d.

P-value

versus

21%

O2

Mea

n±s.d.

P-value

versus

21%

O2

V(lu

ng)[cm

3]

0.26

80±0.01

650.24

48±0.03

400.73

230.26

20±0.03

720.99

910.25

57±0.02

390.97

500.21

64±0.00

900.05

030.26

26±0.02

620.99

94CV[V

(lung

)]0.06

160.14

140.14

210.09

350.04

150.09

97VV(par/lu

ng)[%

]90

.83±

2.30

491

.05±

2.97

3>0.99

9990

.40±

3.40

10.99

9988

.54±

1.91

70.78

7886

.91±

2.70

10.27

1591

.78±

3.25

60.99

41N

(alv,lun

g)(×10

6)

3.99

8±0.43

023.41

8±0.53

200.32

423.37

2±0.73

810.24

912.72

3±0.22

820.00

141.70

1±0.08

90<0.00

011.25

2±0.28

25<0.00

01NV(alv/par)(×10

7)[cm

−3]

1.64

1±0.10

311.53

2±0.04

850.38

791.41

3±0.13

590.00

451.20

6±0.07

21<0.00

010.90

62±0.05

69<0.00

010.51

62±0.07

43<0.00

01CV[N

(alv/lu

ng)]

0.10

760.15

560.21

890.08

380.05

230.22

56SV[cm

−1]

795.9±

33.88

798.9±

23.40

>0.99

9982

0.1±

59.95

0.91

6175

1.0±

38.72

0.46

3760

5.4±

40.95

<0.00

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5.9±

22.01

<0.00

01S(alvep

i,lung

)[cm

2]

193.9±

17.26

177.9±

24.43

0.85

1719

5.1±

38.20

>0.99

9916

9.7±

15.76

0.51

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10.41

<0.00

0112

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12.58

0.00

03CV[S

(alvep

i,lung

)]0.08

900.13

730.19

580.09

290.09

140.10

33V(alv

air,lung

)[cm

3]

0.15

03±0.00

780.12

85±0.02

540.43

990.14

76±0.02

710.99

990.13

00±0.02

080.51

650.11

47±0.00

370.05

310.16

76±0.01

190.67

53τ(sep

)[µm]

9.61

2±0.49

2110

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±0.57

280.25

039.25

5±0.96

320.97

3411

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±0.82

550.00

9812

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±0.70

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0.85

980.00

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120.05

360.10

410.07

220.05

470.07

18MLI

[µm]

31.11±

1.42

828

.73±

1.94

10.82

6230

.48±

3.75

00.99

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2.78

70.99

9640

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32

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191

RESOURCE ARTICLE Disease Models & Mechanisms (2017) 10, 185-196 doi:10.1242/dmm.027086

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generated the most dramatic impact on the lung architecture. Bothprotocols recapitulated the two key pathological hallmarks of BPD:blunted secondary septation (revealed by a decreased alveolarnumber) and an increased mean septal wall thickness; with a morepronounced effect on alveolar number (Fig. 3M), alveolar density(Fig. 3N), andMLI (Fig. 3P) in the P1-P14 85%O2-exposure group,suggesting that these two protocols best model BPD in mice.Although the continuous exposure to 85% O2 from P1 to P14 is aconstant injurious insult, the impact of exposure to 85% O2 from P1to P7, followed by 21% O2 between P7 and P14 is harder to define,as the latter protocol represents a period of injury, followed by aperiod where the lung might repair itself during room-air exposure.This might account for the less severe damage noted in the P1-P785% O2 group. Alternatively, lungs in this group might simply havebeen less damaged as a result of the reduced time frame of exposure

to the injurious insult. Although the degree of damage to thedeveloping lung in these two groups is largely comparable, thecontinuous exposure protocol (P1-P14) is proposed as the method ofchoice, as the lung repair mechanisms that might be engaged afterreturn to 21% O2 in the P1-P7 exposure group representconfounding variables that are not present in the continuousexposure protocol (P1-P14), although it might be argued that that‘oxygen recovery’ period is of translational significance. This‘repair phase’ confounding variable might make data interpretationdifficult, either when dissecting pathogenic pathways modulated byoxygen injury or when investigating the efficacy of a candidate drugto limit damage from oxygen injury. Of additional interest is thelong-term sequelae of hyperoxia exposure in the neonatal period,which remains of interest to model as alveolarisation in humanscontinues into the teen years at least. However, the persistence of

Fig. 4. Comparison of the efficacy of a candidate therapeutic intervention in a severe versus a less severe hyperoxia-based model ofbronchopulmonary dysplasia. Mouse pups within each of the three oxygen exposure groups [continuous ambient (21% O2) room air; 60% O2, or 85% O2;presented in rows] were either sham-treated (control) or treated with cottonseed oil (average of 12 ml kg−1 day−1; see Materials and Methods for precise dosingprotocol) via daily intraperitoneal injection, to provide supplementary parenteral nutrition. (A-L) Images are sections from lungs embedded in glycol methylacrylateplastic after fixation with buffered paraformaldehyde/glutaraldehyde and treatment with sodium cacodylate, osmium tetroxide, and uranyl acetate, then stainedwith Richardson’s stain. Each image is representative of lung sections obtained from four other mouse pups within each experimental group (n=5, per group).Within each treatment group (control versus cottonseed oil), the column of images to the left represents low-magnification images, with higher-magnificationimages derived from part (demarcated by the red box) of the corresponding images presented to the right, to highlight changes in alveolar septal wall thickness.Scale bars: 200 µm. (M-Q) Design-based stereology was employed to assess (M) total number of alveoli in the lung, (N) alveolar density, (O) gas exchangesurface area, (P) mean linear intercept, and (Q) alveolar septal wall thickness. (R) The lung volume was estimated by the Cavalieri method. In panels G-L,• denotesmale animals,▴ denotes female animals. Data represented asmean±s.d. Data comparisons weremade by one-way ANOVAwith Tukey’s post hoc test.Significant P-values are indicated in the graphs; n.s., not significant (P≥0.05). CSO, cottonseed oil-treated group; Ctrl., control (sham-treated) group.

192

RESOURCE ARTICLE Disease Models & Mechanisms (2017) 10, 185-196 doi:10.1242/dmm.027086

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alveolarisation defects beyond P14 in mice was not considered inthis study.

The P5.5 time point is believed to represent the peak of secondaryseptation (Herriges and Morrisey, 2014; Hogan et al., 2014), whichis the driver of alveologenesis. Therefore, mouse pups were alsoexposed to 85%O2 over the period P4-P7 (Fig. 3D,J), believed to bea critical window of secondary septation. Mouse pups were alsoexposed to 85% O2 over the preceding period, P1-P3 (Fig. 3C,I).For both number of alveoli (Fig. 3M) and the mean septal wallthickness (Fig. 3Q), a dramatic impact was noted for the P4-P7exposure periods, but not for the P1-P3 exposure period. These datamight confirm that the period P4-P7 is a critical window of lungdevelopment that might be severely impacted by hyperoxiaexposure; however, it is also noteworthy that the cumulativeoxygen dose for the period P4-P7 is greater (as this period is 24 hlonger) than the P1-P3 period.

It is important to note that the only readout used in this study wasa change in lung structure, where interventions were examined forthe ability to drive changes to the total number of alveoli in the lungand in the thickness of the alveolar septal wall. In selected instances,for example, exposure of newborn mouse lungs to hyperoxia overthe first three days of life, no impact of an oxygen-exposure protocolon lung structure was noted (Fig. 1B, Fig. 3C,I). However, it mightwell be that other important changes do occur in the lung that are ofphysiological and pathological relevance, but which are not evidentfrom an examination of the lung structure alone. Indeed, exposure ofnewborn mice to hyperoxia over the first three days of postnatal lifehas a dramatic impact on the airway hyper-responsiveness in thelong-term (at days 55-77) in adult mice (Regal et al., 2014).Similarly, in the same model, an impact of early exposure tohyperoxia over the first three days of postnatal life was alsodocumented to de-regulate the expression of epithelial (Sftpc,Abca3, Pdpn, Aqp5) as well as endothelial (Pecam) genes (Yeeet al., 2014). Additionally, after slightly longer exposures (for thefirst four days of life) to hyperoxia, persistent changes in naturalkiller cell responses to influenza virus infection were observed inadult life (Reilly et al., 2015). It should be noted that these threestudies employed 100% O2 as an injurious stimulus, but thesereports do indicate that although alterations to lung structure werenot noted after hyperoxia exposure for the first three days ofpostnatal life in our standardised BPD model presented here, otherphysiological changes clearly do occur in the lung.

A key concern in the use of experimental animal models ofhuman disease is that often the most injurious stimulus is employedin an effort to obtain a clearly evident pathological change in aparameter of interest. In these instances, changes are dramatic andparameters are better clustered, yielding a more favourable statisticalcomparison with controls. In the case of the hyperoxia-based mousemodel of BPD, the continuous exposure of newborn mouse pups to85% O2 from P1 to P14 yielded the most pronounced impact on thegas exchange structure (cumulatively reflected by a change in totalnumber of alveoli, alveolar density and gas exchange surface area;Fig. 2M-O) as well as disturbances to alveolar septal wall thickness(Fig. 2Q). However, one danger of this severe model is that a highlyinjurious stimulus might exert such a damaging effect that thepositive (but moderate or weak) impact of a candidate interventionmight go unnoticed. Therefore, this idea was tested by providingnutritional supplementation to newborn mouse pups (in the form ofcottonseed oil) over the course of oxygen exposure between P1 andP14, at both 60% O2 and 85% O2, and examined the magnitude ofimprovement in lung structure in both oxygen-exposure protocols.Indeed, a beneficial impact of cottonseed oil administration wasTa

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60%

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Con

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Cottons

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Con

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Cottons

eedoil

Con

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Cottons

eedoil

Param

eter

Mea

n±s.d.

Mea

n±s.d.

P-value

versus

21%

O2

Mea

n±s.d.

P-value

versus

21%

O2

Mea

n±s.d.

P-value

versus

21%

O2

Mea

n±s.d.

P-value

versus

21%

O2

Mea

n±s.d.

P-value

versus

21%

O2

V(lu

ng)[cm

3]

0.26

46±0.01

440.23

38±0.02

660.56

820.22

26±0.03

800.24

620.29

50±0.01

590.58

150.27

34±0.03

310.99

660.27

56±0.03

810.99

05CV[V

(lung

)]0.05

420.01

138

0.17

070.05

380.12

100.13

81VV(par/lu

ng)[%

]92

.49±

2.31

188

.59±

3.09

70.15

1888

.71±

3.12

50.17

4588

.39±

1.60

20.11

8892

.24±

1.07

4>0.99

9990

.16±

2.63

50.65

64N

(alv,lun

g)(×10

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4.06

7±0.71

053.23

2±0.63

140.08

802.05

8±0.41

49<0.00

013.65

3±0.27

920.72

811.53

6±0.35

34<0.00

011.99

3±0.19

18<0.00

01NV(alv/par)(×10

7)[cm

−3]

1.65

9±0.23

241.58

9±0.13

740.96

711.07

5±0.09

35<0.00

011.40

5±0.13

320.08

450.60

54±0.09

34<0.00

010.81

10±0.11

20<0.00

01CV[N

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0.17

470.19

540.20

160.07

640.23

010.09

62SV[cm

−1]

824.3±

43.12

890.3±

25.74

0.30

1669

3.7±

76.37

0.00

3580

7.7±

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0.99

4048

6.3±

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01S(alvep

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201.7±

18.41

184.7±

24.80

0.65

9713

5.8±

19.28

<0.00

0121

0.6±

12.21

0.96

5112

4.2±

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<0.00

0114

9.1±

8.77

0.00

12CV[S

(alvep

i,lung

)]0.09

130.13

430.14

200.05

800.09

140.10

33V(alv

air,lung

)[cm

3]

0.15

95±0.01

150.13

70±0.02

240.63

920.13

57±0.02

680.58

560.17

62±0.00

810.85

350.18

85±0.01

880.37

240.18

75±0.03

740.40

89τ(sep

)[µm]

8.45

0±0.59

617.28

1±0.89

760.13

388.32

4±0.84

990.99

978.01

6±0.36

760.92

3910

.27±

0.79

220.00

558.22

9±0.60

410.99

60CV[τ(sep

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050.12

330.10

210.04

590.07

710.07

34MLI

[µm]

31.73±

2.26

230

.04±

2.32

00.99

8741

.59±

7.98

30.07

7133

.49±

0.65

10.99

5061

.91±

4.76

0<0.00

0150

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8.81

00.00

02CV[M

LI]

0.07

130.07

630.19

190.01

940.07

690.17

54

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noted in the 60% O2 exposure protocol, but not in the 85% O2

exposure protocol. Thus, if the 85% O2 model alone had beenemployed, the potential utility of cottonseed oil as an intervention inBPD would have been missed. The selection of the oxygen exposureis, therefore, crucially important; and these observations underscorethe idea that if an experimental intervention proves unsuccessful insevere models of disease, a less severe model might neverthelesshighlight the potential utility of the intervention. Further to this,however, the severe (85% O2) BPD model is the only model thatrecapitulates both the disturbances to the gas exchange structure(cumulatively reflected by a change in total number of alveoli,alveolar density and gas exchange surface area; Fig. 2M-P,R) as wellas disturbances to alveolar septal wall thickness that are seen inhuman disease (Fig. 2Q). Although an effect of cottonseed oiladministration on alveoli number in the 85%O2 model was missed, apronounced positive impact of cottonseed oil on alveolar septal wallthickness was still noted in the 85% O2 model, where cottonseed oilapplication normalised the alveolar wall thickness (Fig. 4K,L, versusFig. 4I,J; Fig. 4Q). Taken together, these data reveal that the oxygenexposure protocol requires tailoring and broad consideration whenassessing the potential utility of an intervention intended to drivepostnatal lungmaturation in an experimental animal model of BPD. Itis the recommendation of the authors to employ 85% O2 to generatean impact of hyperoxia on both the number of alveoli and the meanseptal wall thickness. However, when an experimental interventionfails to blunt the impact of 85% O2 on these two parameters of lungstructure, it is recommended to evaluate the same intervention using60% O2 as an injurious stimulus. Keeping animal welfare in mind(Curzer et al., 2016), this model represents the best compromisebetween minimising the number of experimental animals employedand the level of stress to which the animal is subjected by keepingthe injurious insult as mild as possible, while still retaining thetranslational viability of the animal model.This report exclusively addresses the use of hyperoxia as an

injurious stimulus in the arrested lung development associated withhyperoxia exposure, as a model for BPD. Clinically, hyperoxia isone of many contributors to BPD, with additional important diseasemodifiers including, inter alia baro- and volu-trauma frommechanical ventilation (Greenough et al., 2008), and thebackground of infection (Balany and Bhandari, 2015). Futureefforts to further optimise the mouse (and other) models of BPDshould address hyperoxia in combination with mechanicalventilation and/or infection.

MATERIALS AND METHODSApprovals for studies with experimental animalsAll animal procedures were approved by the local authorities, theRegierungspräsidium Darmstadt (approval numbers B2/344, B2/1051,and B2/1108).

Mouse models of bronchopulmonary dysplasiaThe normobaric hyperoxia-based model of BPD in mice was conductedessentially as described previously, with the modifications outlined below(Alejandre-Alcázar et al., 2007; Madurga et al., 2014; Mižíková et al.,2015). Newborn C57Bl/6 mice (Mus musculus Linnaeus) were randomisedto equal-sized litters (average seven mice per litter), and placed into either anormoxic or hyperoxic environment within two hours of birth. Thehyperoxia-exposure protocols are illustrated in Fig. 1A,B. For the 40% O2,60%O2, and 85%O2 oxygen exposure protocols, mouse pups were exposedto the appropriate oxygen concentration starting on the day of birth (P1),continuously up to and including P14. Two additional oxygen levelprotocols were also performed: (1) a decreasing gradient of O2 from 85% onP1 to 21% on P14 (a reduction in oxygen concentration of 5% per day); and

(2) an oscillation between 85% O2 and 40% O2, for a 24 h period, on a24 h:24 h oscillation cycle.

To determine the necessary window of oxygen exposure over the first14 days of life, newborn mouse pups were exposed to 85% O2 for discrete‘windows’, which included: (1) the first 24 h of life (P1), (2) the first threedays of life, starting at P1, up to and including P3; (3) starting at thebeginning of P4 and continuing to (and including) P7; (4) the first sevendays of life, starting at P1, and continuing to (and including) P7; and (5) theentire first 14 days of life, ending with (and including) P14. All experimentswere terminated at P14.

For all oxygen-exposure protocols, nursing dams were rotated every 24 h,to ensure at least one 24 h period of 21% O2 every 2 days. This addresses theoxygen toxicity issues in adult mice, which are highly susceptible toprolonged periods of hyperoxia. Nursing dams received food ad libitum.Mice were maintained in a 12 h:12 h dark/light cycle. All pups wereeuthanised at the end of P14 with an overdose of pentobarbital (500 mg/kg,intraperitoneal; Euthoadorm, CP-Pharma, Burgdorf, Germany), followedby thoracotomy, then by lung extraction and processing for design-basedstereology (Madurga et al., 2014; Mižíková et al., 2015).

Design-based stereologyAll methods employed for the analysis of lung structure were based onAmerican Thoracic Society and European Respiratory Societyrecommendations for quantitative assessment of lung structure (Hsiaet al., 2010). The protocol employed for the design-based stereologicalanalysis of neonatal mouse lungs has been described in the detail previously(Madurga et al., 2015, 2014;Mižíková et al., 2015), based on state-of-the-artmethodology (Mühlfeld et al., 2015, 2013; Mühlfeld and Ochs, 2014, 2013;Ochs and Mühlfeld, 2013; Schneider and Ochs, 2013; Schneider and Ochs,2014). Briefly, mouse lungs were instillation-fixed through a trachealcannula at a hydrostatic pressure of 20 cm H2O with 1.5% (w/v)paraformaldehyde (Sigma, Darmstadt, Germany; P6148), 1.5% (w/v)glutaraldehyde (Serva, Heidelberg; 23116.02) in 150 mM HEPES(Sigma; H0887), pH 7.4, for 24 h at 4°C, after which lung tissue blockswere collected according to systematic uniform random sampling forstereological analysis (Schneider and Ochs, 2014).

Lungs were embedded in toto in 2% (w/v) agar (Sigma; 05039) and cutinto 3 mm sections. The total volume of the lungs was measured byCavalieri’s principle (Madurga et al., 2015, 2014; Mižíková et al., 2015).Whole lungs were treated with sodium cacodylate (Serva; 15540.03),osmium tetroxide (Roth, Karlsruhe, Germany; 8371.3), and uranyl acetate(Serva; 77870.01) and embedded in glycol methacrylate (Technovit 7100;Heareus Kulzer, Hanau, Germany; 64709003). For the determination ofalveoli number, each tissue block was cut into sections of 2 µm, and everyfirst and third section of a consecutive series of sections throughout theblock was stained with Richardson’s stain. For all other parameters, everytenth section of a consecutive series throughout the block was similarlyprepared (four sections per block were selected).

All slides were scanned using a NanoZoomer-XR C12000 Digital slidescanner (Hamamatsu, Herrsching am Ammersee, Germany). Analyses wereperformed using the Visiopharm NewCast computer-assisted stereologysystem (Visiopharm, Hoersholm, Denmark). Parameters analysed includedthe mean linear intercept (MLI), alveolar septal wall thickness, total surfacearea, as well as alveolar number and alveolar density, as described previously(Madurga et al., 2015, 2014;Mižíková et al., 2015). Intrinsic to this analysis isthe separate scoring of parenchymal elements, which are discriminated fromvessels and airways. A total of∼40 tissue sections were evaluated per animal,for all parameters, except the determination of alveolar number, in which casea total of 10 sections per animal were evaluated. In each case, 2-5% of eachsection was analysed. The coefficient of error (CE), the coefficient ofvariation (CV), as well as the squared ratio between both (CE2/CV2) weremeasured for each stereological parameter (Tables 1,2,3), and the quotientthreshold was set at 0.5 to validate the precision of the measurements.

Therapeutic intervention with generic parenteral nutritionCottonseed oil (Sigma-Aldrich, Darmstadt, Germany; C7767) was appliedby daily intraperitoneal injection to pups, where the cottonseed oil dose wasde-escalated over a range starting at 20 ml kg−1 day−1 (P1, P2, P3), followed

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by 15 ml kg−1 day−1 (P4, P5, P6), then by 10 ml kg−1 day−1 (P7, P8, P9)and finally to 5 ml kg−1 day−1 (P10, P11, P12, and P13), to avoid theinjection of a large oil bolus. The experiment was terminated at P14. Thecottonseed oil application was undertaken in the BPD model using eithercontinuous exposure to 60% O2 (FiO2 0.60) or 85% O2 (FiO2 0.85). Theapplication of cottonseed oil was in analogy with generic oil-basednutritional supplementation that is provided to preterm infants with or at riskfor BPD in a neonatal intensive care setting (Beken et al., 2014).

Sex genotyping of miceSex determination of mouse pups was undertaken exactly as describedpreviously (Lambert et al., 2000). Essentially, genomic DNA was isolatedfrom tail biopsies, and screened by polymerase chain reaction using forwardprimer 5′-TGGGACTGGTGACAATTGTC-3′ and reverse primer 5′-GAGTACAGGTGTGCAGCTCT-3′ to detect the male-specific Sry locus;together with forward primer 5′-GGGACTCCAAGCTTCAATCA-3′ andreverse primer 5′-TGGAGGAGGAAGAAAAGCAA-3′ to detect the Il3gene present in both the male and female sex. Amplicons were resolved on a1.5% (w/v) agarose gel, and visualised by ethidium bromide staining.

Statistical analysisAll data are presented as mean±s.d. Differences between groups wereevaluated by one-way ANOVA with Tukey’s post hoc test for allexperiments. P-values <0.05 were regarded as significant. All statisticalanalyses were performed with GraphPad Prism 6.0. The presence ofstatistical outliers was tested by Grubbs’ test, and none were found. All datasets are small for proper determination of normal distribution; however, acorrected Anderson–Darling statistic demonstrated normal distribution ofdata sets.

AcknowledgementsWe thank Dr Martin Post (Hospital for Sick Children, Toronto, Canada), Dr GerryT. M. Wagenaar (Leiden University Medical Center, Leiden, The Netherlands) andDr Christopher D. Baker (University of Colorado Medical Center, Aurora, USA) forhelpful discussions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsC.N., I.M., D.M.G., J.R.-C. and R.E.M.: designed the research; C.N., I.M., D.M.G.and J.R.-C.: performed the hyperoxia studies, tissue extraction and embedding, anddesign-based stereology studies; K.M., I.V., S.H. and W.S.: tissue processing andmicroscopy, assisted in experimental design and data interpretation. C.N. and I.M.prepared the figures. C.N., I.M. and R.E.M. wrote and edited the manuscript.

FundingThis study was financially supported by the Max-Planck-Gesellschaft (Max PlanckSociety); Rhon Klinikum AG (grant Fl_66); the Hessisches Ministerium furWissenschaft und Kunst (Federal Ministry of Higher Education, Research and theArts of the State of Hessen) “LOEWE Programme”, Deutsches Zentrum furLungenforschung (the German Center for Lung Research), and by the DeutscheForschungsgemeinschaft (German Research Foundation) through ExcellenceCluster EXC147, Collaborative Research Center SFB1213/1, Clinical Research UnitKFO309/1, and individual research grant 1789/1.

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