Micrometer-resolution X-ray tomographic full-volume reconstruction of an intact post-mortem juvenile rat lung

Borisova, Elena; Lovrić, Goran; Miettinen, Arttu; Fardin, Luca; Bayat, Sam; Larsson, Anders; Stampanoni, Marco; Schittny, Johannes C.

doi:10.1007/s00418-020-01868-8

Cited by 28 publications

(26 citation statements)

References 57 publications

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“…Own preliminary data point to the same result. We imaged an entire rat lung in situ immediately post mortem using high-resolution synchrotron radiation based X-ray tomographic microscopy [44]. A rst analysis of regional di erence shows similar results as mentioned above.…”

Section: Discussionmentioning

confidence: 59%

Pulmonary acini exhibit complex changes during postnatal rat lung development

Haberthür

Yao

Barré

et al. 2019

Preprint

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1Pulmonary acini represent the functional gas-exchanging units of the lung and are defined as the airways distal of the 2 terminal bronchioles. Due to technical limitations, it was not possible to identify individual acini on microscopic lung 3 sections. Recently, X-ray tomographic microscopy has been adapted to lung imaging to overcome these limitations. This 4 study aimed to follow the acinar development and to gain insight into changes of the acinar structures during postnatal 5 development. Rat lungs were fixed by instillation at postnatal days P4, P10, P21, and P60. Right lower lobes were imaged 6 at the TOMCAT beamline of the Swiss Light Source synchrotron facility at a voxel size of 1.48 µm. Individual acini were 7 segmented out of the obtained 3D data sets by closing the airways at the acinar entrance which is the transition point 8 from conducting to gas exchanging airways happens. Acinar development was followed by estimating the individual acinar 9 volume, complexity, and number of alveoli (N=267). We observed that the mean volume of the acini increases 10 times 10 between days 4 and 60 which corresponds to a 10 times increase of the total lung volume. During the same time, the 11 coefficients of variation dropped from 1.26 to 0.49 and the difference between the mean volumes of the fraction of the 20 % 12 smallest and 20 % largest acini decreased from a factor of 27.26 (day 4) to a factor of 4.07 (day 60). This indicates that the 13 dispersion of the value is based on an entirely different pattern of distribution of acinar sizes. Methodologically, we conclude 14 that our approach is well suited to follow acinar development and to acquire large amounts of data comparable to classical 15 stereology in a time-efficient manner. Biologically, we conclude that the newly formed acini show a large size distribution 16 which is reduced by a factor of 7 until adulthood. Most likely the latter leads to a more homogeneous ventilation of the 17 lung. 18 1 Introduction 19 1.1 Lung development 20Lung development starts with the formation of the two lung buds. During the embryonic stage (prenatal 21 days E11-E13 in rats) the major airways and the pleura are formed. During the pseudoglandular stage 22 (E13-E18.5) most of the remaining airway generations are formed by branching morphogenesis. Few 23 additional branches are added during the canalicular (E18.5 -E20) and maybe the saccular stage (E20 24 to postnatal day P4). Epithelial differentiation becomes morphologically visible during the canalicular 25 stage. Together with angiogenetic activation of the capillaries, this process leads to the first functional 26 air-blood barriers in the lung. 27The saccular stage represents an intermediate stage where branching morphogenesis ceases and the 28 developmental program switches to the phase of alveolarization. During alveolarization, 90 % of the adult 29 gas-exchange area is formed by the lifting off of new septa from the existing gas-exchange surface (Schittny 30 2017). The alveolarization itself is divided into two d...

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Section: Discussionmentioning

confidence: 59%

Pulmonary acini exhibit complex changes during postnatal rat lung development

Haberthür

Yao

Barré

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Own preliminary data point to the same result. We imaged an entire rat lung in situ immediately post mortem using high-resolution synchrotron radiation based X-ray tomographic microscopy [ 47 ]. A first analysis of regional difference shows similar results as mentioned above.…”

Section: Discussionmentioning

confidence: 99%

Pulmonary acini exhibit complex changes during postnatal rat lung development

et al. 2021

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Pulmonary acini represent the functional gas-exchanging units of the lung. Due to technical limitations, individual acini cannot be identified on microscopic lung sections. To overcome these limitations, we imaged the right lower lobes of instillation-fixed rat lungs from postnatal days P4, P10, P21, and P60 at the TOMCAT beamline of the Swiss Light Source synchrotron facility at a voxel size of 1.48 μm. Individual acini were segmented from the three-dimensional data by closing the airways at the transition from conducting to gas exchanging airways. For a subset of acini (N = 268), we followed the acinar development by stereologically assessing their volume and their number of alveoli. We found that the mean volume of the acini increases 23 times during the observed time-frame. The coefficients of variation dropped from 1.26 to 0.49 and the difference between the mean volumes of the fraction of the 20% smallest to the 20% largest acini decreased from a factor of 27.26 (day 4) to a factor of 4.07 (day 60), i.e. shows a smaller dispersion at later time points. The acinar volumes show a large variation early in lung development and homogenize during maturation of the lung by reducing their size distribution by a factor of 7 until adulthood. The homogenization of the acinar sizes hints at an optimization of the gas-exchange region in the lungs of adult animals and that acini of different size are not evenly distributed in the lungs. This likely leads to more homogeneous ventilation at later stages in lung development.

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“…The image on the scintillator was magnified 4 times using a high numerical aperture macroscope custom built by Optique Peter (Lentilly, France) [ 24 ] and captured using a pco.EDGE 5.5 (PCO AG, Kelheim, Germany) sCMOS camera, resulting in an effective isometric pixel size of 1.625 μm. 3–7 wide-field 360-degree scans were stacked vertically with slight overlaps along the rotational axis [ 25 – 27 ] for each sample to capture its complete volume. Tomographic reconstructions of the individual 3D-datasets were performed in single-distance phase contrast mode [ 28 ] (δ = 2e-7, β = 2.8e-10) with the gridrec algorithm [ 29 ] using the TOMCAT reconstruction pipeline (Reco Manager) [ 30 ], and finally stitched together using the non-rigid stitching algorithm NRStitcher [ 31 ].…”

Section: Methodsmentioning

confidence: 99%

Early life exposure to nicotine modifies lung gene response after elastase-induced emphysema

et al. 2022

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Background Chronic obstructive pulmonary disease (COPD) is among the top 5 causes of mortality in the world and can develop as a consequence of genetic and/or environmental factors. Current efforts are focused on identifying early life insults and how these contribute to COPD development. In line with this, our study focuses on the influence of early life nicotine exposure and its potential impact on (a) lung pulmonary functions, and (b) elastase-induced emphysema in adulthood. Methods To address this hypothesis, we developed a model of 2 hits, delivered at different time points: mouse pups were first exposed to nicotine/placebo in utero and during lactation, and then subsequently received elastase/placebo at the age of 11 weeks. The effect of nicotine pretreatment and elastase instillation was assessed by (a) measurement of pulmonary function at post-elastase day (ped) 21, and (b) transcriptomic profiling at ped3 and 21, and complementary protein determination. Statistical significance was determined by 3- and 2-way ANOVA for pulmonary functions, and RNAseq results were analyzed using the R project. Results We did not observe any impact of nicotine pre- and early post-natal exposure compared to control samples on lung pulmonary functions in adulthood, as measured by FLEXIVENT technology. After elastase instillation, substantial lung damage was detected by x-ray tomography and was accompanied by loss in body weight at ped3 as well as an increase in cell numbers, inflammatory markers in BAL and lung volume at ped21. Lung functions showed a decrease in elastance and an increase in deep inflation volume and pressure volume (pv) loop area in animals with emphysema at ped21. Nicotine had no effect on elastance and deep inflation volume, but did affect the pv loop area in animals with emphysema at ped21. Extensive transcriptomic changes were induced by elastase at ped3 both in the nicotine-pretreated and the control samples, with several pathways common to both groups, such as for cell cycle, DNA adhesion and DNA damage. Nicotine pretreatment affected the number of lymphocytes present in BAL after elastase instillation and some of the complement pathway related proteins, arguing for a slight modification of the immune response, as well as changes related to general body metabolism. The majority of elastase-induced transcriptomic changes detected at ped3 had disappeared at ped21. In addition, transcriptomic profiling singled out a common gene pool that was independently activated by nicotine and elastase. Conclusions Our study reports a broad spectrum of transient transcriptomic changes in mouse emphysema and identifies nicotine as influencing the emphysema-associated immune system response.

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Micrometer-resolution X-ray tomographic full-volume reconstruction of an intact post-mortem juvenile rat lung

Cited by 28 publications

References 57 publications

Pulmonary acini exhibit complex changes during postnatal rat lung development

Pulmonary acini exhibit complex changes during postnatal rat lung development

Pulmonary acini exhibit complex changes during postnatal rat lung development

Early life exposure to nicotine modifies lung gene response after elastase-induced emphysema

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