Scanning Electron Microscopy of Styrene-Methylethylketone Casts of the Airway and the Arterial System of the Lung

The method of making casts of airways and the pulmonary arterial system using a styrene polymer resin is described. A human and a dog were used as models. The viscosity of this resin ranges between 40 and 60 dPa/s at 24°C. The elastic rubber tube used for resin injection allows a constant perfusion pressure, i.e., 44 mm Hg and 110 mm Hg for airway and vascular filling, respectively. The casts obtained using this method are suitable both for macro- and microscopic analysis. Shrinkage during polymerization is minimal. Moreover, a preceding lavage is not necessary. Scanning electron microscopy (SEM) of cast lungs showed the endothelial cell nuclei imprints on vessels. Computer assisted analysis of alveolar capillary interspaces showed that these holes are larger than those described in normal rats. The diameter of alveolar capillaries in dogs is almost the same as in the normal rat lung. When casting the airways, alveolar cells and/or materials inhaled might be preserved and observed in SEM

Fractal analysis reveals functional unit of ventilation in the lung

Ventilation is inhomogeneous in the lungs across species. It has been hypothesized that ventilation inhomogeneity is largely determined by the design of the airway branching network. Because exchange of gases at the alveolar barrier is more efficient when gas concentrations are evenly distributed at subacinar length scales, it is assumed that a 'functional unit' of ventilation exists within the lung periphery, where gas concentration becomes uniform. On the other hand, because the morphology of pulmonary airways and alveoli, and the distribution of inhaled fluorescent particles show self-similar fractal properties over a wide range of length scales, it has been predicted that fractal dimension of ventilation approaches unity within an internally homogeneous functional unit of ventilation. However, the existence of such a functional unit has never been demonstrated experimentally due to lack of in situ gas concentration measurements of sufficient spatial resolution in the periphery of a complex bifurcating network. Here, using energy-subtractive synchrotron radiation tomography, we measured the distribution of an inert gas (Xe) in the in vivo rabbit lung during Xe wash-in breathing manoeuvres. The effects of convective flow rate, diffusion and cardiac motion were also assessed. Fractal analysis of resulting gas concentration and tissue density maps revealed that fractal dimension was always smaller for Xe than for tissue density, and that only for the gas, a length scale existed where fractal dimension approached unity. The length scale where this occurred was seen to correspond to that of a rabbit acinus, the terminal structure comprising only alveolated airways. Key points Gas ventilation is inhomogeneous in the lung of many species. However, it is not known down to what length scales this inhomogeneity persists. It is generally assumed that ventilation becomes homogeneous at subacinar length scales, beyond the spatial resolution of commonly available imaging techniques, hence this has not been demonstrated experimentally. Here we measured the distribution of inhaled Xe gas in the rabbit lung using synchrotron radiation energy-subtractive imaging and used fractal analysis to show that ventilation becomes internally uniform within regions about the size of rabbit lung acini.Peer reviewe

Radiation dose optimized lateral expansion of the field of view in synchrotron radiation X-ray tomographic microscopy

Increasing the lateral field of view of tomography-based imaging methods greatly increases the acquisition time. This article presents scanning protocols to obtain high-resolution tomographic scans with large lateral field of view at greatly decreased acquisition time and thus reduced radiation dose while resulting in high-quality three-dimensional tomographic datasets

Aerosols in Healthy and Emphysematous In Silico Pulmonary Acinar Rat Models

International audienceThere has been relatively little attention given on predicting particle deposition in the respiratory zone of the diseased lungs despite the high prevalence of chronic obstructive pulmonary disease (COPD). Increased alveolar volume and deterioration of alveolar septum, characteristic of emphysema, may alter the amount and location of particle deposition compared to healthy lungs, which is particularly important for toxic or therapeutic aerosols. In an attempt to shed new light on aerosol transport and deposition in emphysematous lungs, we performed numerical simulations in models of healthy and emphysematous acini motivated by recent experimental lobar-level data in rats~\citep{OakesEmp}. Compared to healthy acinar structures, models of emphysematous subacini were created by removing inter-septal alveolar walls and enhancing the alveolar volume in either a homogeneous or heterogeneous fashion. Flow waveforms and particle properties were implemented to match the experimental data. The occurrence of flow separation and recirculation within alveolar cavities was found in proximal generations of the healthy zones, in contrast to the radial-like airflows observed in the diseased regions. In agreement with experimental data, simulations point to particle deposition concentrations that are more heterogeneously distributed in the diseased models compared with the healthy one. Yet, simulations predicted less deposition in the emphysematous models in contrast to some experimental studies, a likely consequence due to the shallower penetration depths and modified flow topologies in disease compared to health. These spatial-temporal particle transport simulations provide new insight on deposition in the emphysematous acini and shed light on experimental observations

Optimal diameter reduction ratio of acinar airways in human lungs

In the airway network of a human lung, the airway diameter gradually decreases through multiple branching. The diameter reduction ratio of the conducting airways that transport gases without gas exchange is 0.79, but this reduction ratio changes to 0.94 in acinar airways beyond transitional bronchioles. While the reduction in the conducting airways was previously rationalized on the basis of Murray's law, our understanding of the design principle behind the acinar airways has been far from clear. Here we elucidate that the change in gas transfer mode is responsible for the transition in the diameter reduction ratio. The oxygen transfer rate per unit surface area is maximized at the observed geometry of acinar airways, which suggests the minimum cost for the construction and maintenance of the acinar airways. The results revitalize and extend the framework of Murray's law over an entire human lung

The total number of acini remains constant throughout postnatal rat lung development.

The pulmonary airways are subdivided into conducting and gas-exchanging airways. The small tree of gas-exchanging airways which is fed by the most distal conducting airway represents an acinus. Very little is known about the development of the number of acini. The goal of this study was to estimate their number throughout rat postnatal development. Right middle rat lung lobes were obtained at postnatal day 4-60, stained with heavy metals, paraffin embedded, and scanned by synchrotron radiation-based X-ray tomographic microscopy or imaged with micro computed tomography after critical point drying. The acini were counted by detection of the transitional bronchioles [bronchioalveolar duct junction (BADJ)] by using morphological criteria (thickness of the walls of airways and appearance of alveoli) during examination of the resulting three-dimensional (3D) image stacks. Between postnatal days 4-60, the number of acini per lung remained constant (5,840 ± 547 acini), but their volume increased significantly. We concluded that the acini are formed before the end of the saccular stage (before postnatal day 4) and that the developmental increase of the lung volume is achieved by an increase of the acinar volume and not by an increase of their number. Furthermore, our results propose that the bronchioalveolar stem cells, which are residing in the BADJ, are as constant in their location as the BADJ itself

Micro-imaging of the Mouse Lung via MRI

Quantitative measurement of lung microstructure is of great significance in assessment of pulmonary disease, particularly in the earliest stages. Conventional stereological assessment of ex-vivo fixed tissue specimens under the microscope has a long and successful tradition and is regarded as a gold standard, but the invasive nature limits its applications and the practicality of use in longitudinal studies. The technique for diffusion MRI-based 3He lung morphometry was previously developed and validated for human lungs, and was recently extended to ex-vivo mouse lungs. The technique yields accurate, quantitative information about the microstructure and geometry of acinar airways. In this dissertation, the 3He lung morphometry technique is for the first time successfully implemented for in-vivo studies of mice. It can generate spatially-resolved maps of parameters that reveal the microstructure of mouse lung. Results in healthy mice indicate excellent agreement between in-vivo morphometry via 3He MRI and microscopic morphometry after sacrifice. The implementation and validation of 3He morphometry in healthy mice open up new avenues for application of the technique as a precise, noninvasive, in-vivo biomarker of changes in lung microstructure, within various mouse models of lung disease. We have applied 3He morphometry to the Sendai mouse model of lung disease. Specifically, the Sendai-virus model of chronic obstructive lung disease has demonstrated an innate immune response in mouse airways that exhibits similarities to the chronic airway inflammation in human COPD and asthma, but the effect on distal lung parenchyma had not been investigated. We imaged the time course and regional distribution of mouse lung microstructural changes in vivo after Sendai virus: SeV) infection with 1H and 3He diffusion MRI. 1H MR images detected the SeV-induced pulmonary inflammation in vivo and 3He lung morphometry showed modest increase in alveolar duct radius distal to airway inflammation, particularly in the lung periphery, indicating airspace enlargement after virus infection. Another important application of the imaging technique is the study of lung regeneration in a pneumonectomy: PNX) model. Partial resection of the lung by unilateral PNX is a robust model of compensatory lung growth. It is typically studied by postmortem morphometry in which longitudinal assessment in the same animal cannot be achieved. Here we successfully assess the microstructural changes and quantify the compensatory lung growth in vivo in the PNX mouse model via 1H and hyperpolarized 3He diffusion MRI. Our results show complete restoration in lung volume and total alveolar number with enlargement of alveolar size, which is consistent with prior histological studies conducted in different animals at various time points. This dissertation demonstrates that 3He lung morphometry has good sensitivity in quantifying small microstructural changes in the mouse lung and can be applied to a variety of mouse pulmonary models. Particularly, it has great potential to become a valuable tool in understanding the time course and the mechanism of lung growth in individual animals and may provide insight into post-natal lung growth and lung regeneration

Morphometrische Analyse des Azinus der humanen Lunge mittels synchrotron-basierter Mikro-Computertomographie

In der vorliegenden Arbeit wurde erstmals ein vollständiger dreidimensionaler Datensatz des Azinus der humanen Lunge mittels der Synchrotron-basierten Mikro-CT-Technik erstellt und anschließend eine quantitative Untersuchung der Strukturen des Azinus durchgeführt. Eine humane Lunge wurde durch die Beatmung mit Formalindampf in Inspirationsstellung fixiert und in einem 64-Zeilen Computertomographen gescannt. Die anschließend ausgestanzten Proben (N = 12, Durchmesser = 8 mm, Höhe = 10 mm) wurden mit Osmiumtetroxid kontrastverstärkt und mit einer Auflösung von 3,9 µm3 Voxelgröße in einem Mikro-CT mit Synchrotronstrahlenquelle gescannt. Mit einem automatisierten „Tree-Analysis“ Softwareprogramm konnten 8 Azini aus dem Volumendatensatz segmentiert werden. Die Morphometrischen Daten wurden mit dem Softwareprogramm ANALYZE 9.0 erhoben. Die Atemwege innerhalb der Azini verzweigten sich über 11 Generationen. Das mittlere Azinusvolumen wurde mit 131,3 ± 29,2 mm3 berechnet und die mittlere Oberfläche des Azinus betrug 1012 ± 26 cm2. Der Innere Durchmesser verringerte sich ausgehend von dem Bronchiolus terminalis von 0,66 ± 0,04 mm auf 0,34 ± 0,06 mm (P < 0,001) und blieb nach der siebten Generation konstant (P < 0,5). Die Länge der einzelnen Generationen variierte zwischen 0,52 und 0,93 mm und zeigte keine signifikanten Unterschiede zwischen der zweiten und elften Generation. Der Verzweigungswinkel der „Tochteräste“ schwankte zwischen 113º und 134º ohne signifikante Unterschiede zwischen den Generationen (P < 0,3). Die in vorangegangenen Arbeiten gezeigten Ergebnisse bezüglich der Morphometrie des humanen Azinus basieren auf den Methoden der Histomorphometrie. Diese arbeitet gewebedestruktiv und stellt lediglich Rückschlüsse von zweidimensionalen Serienschnitten auf dreidimensionale Strukturen her. Unsere Arbeit zeigt die Möglichkeit der quantitativen Erfassung der Strukturen des Azinus anhand eines vollständigen Volumendatensatzes unter Verwendung der Synchrotron- basierten Mikro-CT- Technik.Structural data about the human lung fin structure are mainly based on stereological methods applied to serial sections. As these methods utilize 2D images, which are often not contiguous, they suffer from inaccuracies which are overcome by analysis of 3D micro-CT images of the never-sectioned specimen. The purpose of our study was to generate a complete data set of the intact three-dimensional architecture of the human acinus using high resolution synchrotron-based micro-CT (synMCT). A human lung was inflation-fixed by formaldehyde ventilation and then scanned in a 64-slice CT over its apex to base extent. Lung samples (8-mm diameter, 10 mm height, N = 12) were punched out, stained with osmium tetroxide, and scanned using synMCT at (4 µm)3 voxel size. The lung functional unit (acinus, N = 8) was segmented from the 3D tomographic image using an automated tree-analysis software program. Morphometric data of the lung were analyzed by ANOVA. Intra-acinar airways branching occurred over 11 generations. The mean acinar volume was 131, 3 ± 29, 2 mm3 (range 92, 5- 171, 3 mm3) and the mean acinar surface was calculated with 1012 ± 26 cm2. The airway internal diameter (starting from the bronchiolus terminalis) decreases distally from 0,66 ± 0,04 mm to 0,34 ± 0,06 mm (P < 0,001) and remains constant after the seventh generation (P < 0,5). The length of each generation ranges between 0, 52 and 0, 93 mm and did not show significant differences between the second and eleventh generation. The branching angle between daughter branches varies between 113-degree and 134-degree without significant differences between the generations (P < 0, 3). This study demonstrates the feasibility of quantitating the 3D structure of the human acinus at the spatial resolution readily achievable using synMCT

Keuhkojen rakenteen ja toiminnan kuvantaminen synkrotronisäteilyllä

A novel method for functional lung imaging was introduced by adapting the K-edge subtraction method (KES) to in vivo studies of small animals. In this method two synchrotron radiation energies, which bracket the K-edge of the contrast agent, are used for simultaneous recording of absorption-contrast images. Stable xenon gas is used as the contrast agent, and imaging is performed in projection or computed tomography (CT) mode. Subtraction of the two images yields the distribution of xenon, while removing practically all features due to other structures, and the xenon density can be calculated quantitatively. Because the images are recorded simultaneously, there are no movement artifacts in the subtraction image. Time resolution for a series of CT images is one image/s, which allows functional studies. Voxel size is 0.1mm3, which is an order better than in traditional lung imaging methods. KES imaging technique was used in studies of ventilation distribution and the effects of histamine-induced airway narrowing in healthy, mechanically ventilated, and anaesthetized rabbits. First, the effect of tidal volume on ventilation was studied, and the results show that an increase in tidal volume without an increase in minute ventilation results a proportional increase in regional ventilation. Second, spiral CT was used to quantify the airspace volumes in lungs in normal conditions and after histamine aerosol inhalation, and the results showed large patchy filling defects in peripheral lungs following histamine provocation. Third, the kinetics of proximal and distal airway response to histamine aerosol were examined, and the findings show that the distal airways react immediately to histamine and start to recover, while the reaction and the recovery in proximal airways is slower. Fourth, the fractal dimensions of lungs was studied, and it was found that the fractal dimension is higher at the apical part of the lungs compared to the basal part, indicating structural differences between apical and basal lung level. These results provide new insights to lung function and the effects of drug challenge studies. Nowadays the technique is available at synchrotron radiation facilities, but the compact synchrotron radiation sources are being developed, and in relatively near future the method may be used at hospitals.Tutkimuksen taustaa: Useimmissa keuhkosairauksissa, erityisesti astmassa ja keuhkoahtaumataudissa kaasujen vaihdunta keuhkoissa on häiriytynyt ja keuhkotuuletuksen jakauma on epätasainen. Keuhkojen tuuletuskykyä tutkitaan perinteisesti spirometrialla. Keuhkojen alueellisen toiminnan kuvantamiseen käytetään kliinisesti radionuklidimenetelmiä, joissa erotuskyky on 1-2 senttimetrin luokkaa. Käytössä olevien kuvantamismenetelmien erotuskyky ei ole riittävän tarkka kuvaamaan keuhkojen ääreisosien rakennetta ja toimintaa. Nykyiset menetelmät eivät myöskään ole täysin kvantitatiivisia. Tieto keuhkojen toiminnasta on tähän mennessä rajoittunut alueellisiin suureisiin ja tästä johtuen keuhkojen ääreisosien toiminta on huonosti tunnettu. Tässä väitöskirjatyössä on kehitetty synkrotronisäteilyn käyttöön perustuva uusi menetelmä, jonka avulla keuhkojen ääreisosien toimintaa ja rakennetta voidaan tutkia entistä tarkemmin. Tutkimusmenetelmästä: Synkrotronisäteily on hiukkaskiihdyttimessä tuotettua röntgensäteilyä, jota on perinteisesti käytetty fysiikassa materiaalitutkimuksissa. Synkrotronisäteilyn käyttö lääketieteellisissä tutkimuksissa on suhteellisen uusi tutkimusala, ja yksi menetelmistä on K-reuna vähennyskuvaus (K-edge subtraction imaging, KES). KES-kuvauksessa kuvataan varjoaineen jakautumaa kohteessa käyttäen kahta röntgensäteilyn aallonpituutta, jotka ovat eri puolilla varjoaineena käytettävän stabiilin ksenon-kaasun K-absorptioreunaa. Varjoaineen jakauma keuhkoissa voidaan kuvata CT leikekuvina tai kolmiulotteisesti spiraali-CT:llä, ja hengitystiet sekä keuhkojen ääreisosat saadaan näkyviin. Varjoaineen tiheys voidaan mitata ja sen määrä keuhkoissa on suoraan verrannollinen paikalliseen keuhkotuuletukseen. Kuvien resoluutio on 0.1mm3 ja vertailtuna muihin menetelmiin resoluutio, tarkkuus ja herkkyys tekevät KES-menetelmästä ainutlaatuisen. Tuloksista ja niiden merkityksestä: Väitöskirjatyössä menetelmää on sovellettu keuhkojen rakenteen ja toiminnan yksityiskohtaiseen tutkimiseen ja ymmärtämiseen. Menetelmää on menestyksekkäästi sovellettu keuhkotuuletuksen jakauman tutkimiseen, keuhkojen ja keuhkoputkien kolmiulotteiseen kuvantamiseen ja histamiinilla aiheutetun keuhkoputkien supistumisen dynamiikan tutkimiseen nukutetuilla kaneilla. Tulokset tuovat uutta tietoa keuhkojen toiminnasta ja astmatyyppisistä reaktioista sekä niiden vaikutuksesta keuhkotuuletukseen ja keuhkotilavuuksiin. Toistaiseksi tutkimus voidaan tehdä vain synkrotroni-tutkimuslaitoksissa, mutta kehitteillä on sairaalakäyttöön soveltuvia synkrotronilähteitä; lähitulevaisuudessa menetelmää voitaneen käyttää myös sairaalatutkimuksissa