THE ANATOMICAL RECORD 292:720–727 (2009)

Study of the Variability in Upper andLower Airway Morphology in Sprague–Dawley Rats Using Modern Micro-CTScan-Based Segmentation Techniques

JAN W. DE BACKER,1,3* WIM G. VOS,3 PATRICIA BURNELL,4

STIJN L. VERHULST,3 PHIL SALMON,5

NORA DE CLERCK,6 AND WILFRIED DE BACKER3

1FluidDA, Drie Eikenstraat 661, 2650 Edegem-Antwerp, Belgium2Vision Lab, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk–Antwerp, Belgium

3Department of respiratory medicine, University Hospital Antwerp, Wilrijkstraat 10,2650 Edegem, Antwerp, Belgium

4GlaxoSmithKline Research and Development, Park Road, Ware, Hertfordshire,SG12 0DP, UK

5Skyscan, Kartuizersweg 3B, 2550 Kontich, Belgium6Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1,

2610 Wilrijk–Antwerp, Belgium

ABSTRACTAnimal models are being used extensively in pre-clinical and safety

assessment studies to assess the effectiveness and safety of new chemicalentities and delivery systems. Although never entirely replacing the needfor animal testing, the use of computer simulations could eventuallyreduce the amount of animals needed for research purposes and refinethe data acquired from the animal studies. Computational fluid dynamicsis a powerful tool that makes it possible to simulate flow and particlebehavior in animal or patient-specific respiratory models, for purposes ofinhaled delivery. This tool requires an accurate representation of the re-spiratory system, respiration and dose delivery attributes. The aim of thisstudy is to develop a representative airway model of the Sprague–Dawleyrat using static and dynamic micro-CT scans. The entire respiratory tractwas modeled, from the snout and nares down to the central airways atthe point where no distinction could be made between intraluminal airand the surrounding tissue. For the selection of the representative model,variables such as upper airway movement, segmentation length, airwayvolume and size are taken into account. Dynamic scans of the nostrilregion were used to illustrate the characteristic morphology of this regionin anaesthetized animals. It could be concluded from this study that itwas possible to construct a highly detailed representative model of aSprague–Dawley rat based on imaging modalities such as micro-CT scans.Anat Rec, 292:720–727, 2009. VVC 2009 Wiley-Liss, Inc.

Keywords: small animal imaging; micro-CT; rat; respiratorytract; 3D model

Grant sponsor: GlaxoSmithKline.*Correspondence to: Jan W. De Backer, University Hospital

Antwerp, Department of respiratory medicine, Wilrijkstaat 10,2650 Edegem, Antwerp, Belgium. Fax: þ32 3 821 44 47.E-mail: Jan.DeBacker@ua.ac.be

Received 4 July 2008; Accepted 11 December 2008

DOI 10.1002/ar.20877Published online 25 March 2009 in Wiley InterScience (www.interscience.wiley.com).

VVC 2009 WILEY-LISS, INC.

Animal models are being used extensively in pre-clini-cal and safety assessment studies to assess the effective-ness and safety of new chemical entities, delivery systemsor to investigate the safe levels for occupational exposure(Alfaro et al., 2004; Chapman et al., 2007; Ji et al., 2007).Often small animals, like mice or rats, are exposed to theproduct for a longer periods of time (Wagner et al., 2006;Lee et al., 2007) or for shorter periods of time to assessacute effects (Dong et al., 2005; Kouadio et al., 2005). Asin human trials (De Backer et al., 2007a) a trend isemerging in animal studies towards accurate computermodels describing the animals’ physiological behavior(Schroeter et al., 2006; Galle et al., 2007). Although neverentirely replacing animal studies, this could, eventually,reduce the amount of animals needed for research.

The increasing prevalence of respiratory diseases suchas asthma and chronic obstructive pulmonary disease(COPD) intensified the research regarding the morphol-ogy and physiology of the respiratory system both clini-cally as well as pre-clinically. A tool that is increasinglyused to assess the respiratory function is computationalfluid dynamics or CFD. This method is capable of simu-lating flow behavior in virtual models (Bush et al., 1998;Andersen et al., 2000; Minard et al., 2006). The challengeis to develop mathematical models that realisticallyreflect the respiratory physiology of the animal by accu-rately simulating its fluid flow conditions. These modelscomprise of accurate geometrical representations of theairway structures and realistic boundary conditionswhich drive the flow (De Backer et al., 2007b). The CFDmethod and these models can assist in the developmentof new inhalation therapies and the optimization ofinhaler devices (Coates et al., 2007; Kleinstreuer et al.,2007). Advances in small animal imaging using micro-CTor MRI have made it possible to study the respiratorysystem in a specific animal into great detail (Johnson,2007; Lam et al., 2007; Wietholt et al., 2008). Earlierwork by Chaturvedi and Lee (Chaturvedi and Lee, 2005)has used micro CT images to accurately study the mor-phology of the mouse and canine airway tract based onlung casts and segmentation principles. The study wepresent here uses similar techniques but scans are per-formed in vivo or in situ. Our study aims to develop arepresentative, realistic model of the rat respiratory tractbased on high-resolution micro-CT scans. In particularthe variability between different animals from the samespecies will be investigated as also performed in anotherrecent study by Lee et al. (Lee et al., 2008). Lee et al.again used lung casts to assess the geometrical featuresof the respiratory system of healthy Sprague–Dawleyrats. In our study, a range of techniques (static anddynamic micro-CT scans) will be used in different circum-stances (anaesthetized and euthanized animals) to obtainin vivo and in situ images. Differences between differentrats were assessed to obtain a representative model. Boththe nasal passages and the tracheobronchial tree wereconsidered in the model. The resulting representativemodel could subsequently be used in flow and particledeposition simulation studies.

MATERIALS AND METHODS

In this study a total of 11 Sprague–Dawley rats wereused. The average weight of the animals was 372 � 56 g.Micro-CT scans (Skyscan 1076 high resolution in vivo

micro-CT, Skyscan, Belgium) were taken of the snoutand entire respiratory tract of the rats. The scanner hada tube diameter of 68 mm and a 17 mm single scanlength. For the scans a resolution of 35 lm was selected.An initial test comparing scans from anaesthetized witheuthanized rats clearly showed that resolution of airwaystructures in the euthanized rat was superior to theanaesthetized rat. Therefore, the airways in the lungswere scanned in 7 euthanized rats to optimize the imagequality. The tracheobronchial tree was defined as theairways starting from the larynx down. The nasal pas-sages were scanned in a total of 8 rats. A definition ofthe airway regions is given in Fig. 1 and an overview ofthe scans is provided in Table 1. In addition to the vol-ume of the lower airways, the airway length was deter-mined through the construction of centerlines based onthe segmentation masks

lCT Scanning of the Respiratory System

The first rats (rat 1–4) were scanned from the nostrilsdown to the diaphragm to include the lungs and all extra-thoracic structures (snout, nares, nasopharynx, epiglottis,and trachea) The total scanning length was �12 cm (Fig.1) and required around 6–7 scans per animal. Scans weretaken with a source voltage of 100 kV and a source currentof 140 lA. The resolution was set to 35 lm and the rotationstep was 0.6 degrees. The rats needed to be euthanized forthese scans because the scanning time would have takentoo long (>5 hr) to bring animals under anesthesia and topreserve image quality due to motion artifacts. The ani-mals were euthanized with 1 mL of Nembutal and wereleft for �1 hr to complete the state of rigor mortis. Toincrease the number of animal-specific tracheobronchialmorphologies, only the tracheobronchial regions of 3 addi-tional rats (rats 5–7) were scanned additionally.

Dynamic lCT Scanning of the Nasal Passages

Rats are obligate nasal breathers, using their sense ofsmell to identify and classify objects. Consequently, thenasal passages are well developed and have an impor-tant filtering function to prevent hazardous particlesfrom entering the animals’ respiratory system. To assessthe movement of the nostril and upper airway regionduring breathing, dynamic scans of this region weretaken in 4 rats (rats 8–11) in addition to the static lCTscans of rats 1–4. Scans were taken with a source volt-age of 70 kV and a source current of 140 lA. The resolu-tion was set to 35 lm and the rotation step was 0.9degrees. The scan length was �1.5 cm. The rats wereanaesthetized and their breathing pattern was moni-tored. The dynamic scans were performed using respira-tory gating. The animals were anaesthetized with a 50-50 mixture of Nembutal and saline. The dosage wasbased on 35 mg of Nembutal per 2.5 kg of bodyweight.The respiratory gating was based on a visual triggeringsystem. The motion of the animal’s thorax was gatedand whenever a clear signal was detected four imageswere taken and the registration time was recorded (Fig.2). Afterwards all images were sorted into several binsto attain static images at certain breathing levels.Therefore images of the entire region of interest whereobtained for the normal inspiration and expiration.

AIRWAY MORPHOLOGY IN SPRAGUE–DAWLEY RATS 721

Combining these static images produced a dynamicimage, indicating the changes over time.

Airway Segmentation

All scans from both anaesthetized and euthanized ani-mals were then read into a commercially available andvalidated software package Mimics (Materialise, Leuven,Belgium). Three-dimensional reconstructions of theupper and lower airways were made, based on segmen-tation principles. Voxels with Hounsfield Units, a mea-sure of electron density, within the pre-described range(�1,024 to �865) were placed in a separate mask. Athree-dimensional virtual representation of the structurewas reconstructed from this mask. Airway lengths, sur-faces, and volumes could be measured from this recon-struction. The selection process for the ‘average animalmodel’ is explained in the results and discussion section.Segmentation into the various lung lobes was carriedout using the ‘average model’. This was done by seg-menting the lung volumes and subsequently defining thefissures separating the lung lobes.

Ethical Approval

This study has been approved by the ethical commit-tee of the University of Antwerp. Animals were handled

by the qualified staff of the animalarium of the Univer-sity of Antwerp.

RESULTSLower Airway

A visual representation of the reconstructed lower air-ways of rat 1–7 can be found in Fig. 3. The typical struc-ture of the airway tree can be clearly seen in Fig. 3.Measurements of the segmented lower airway volumegave an arithmetic mean airway volume and standarddeviation of 332.40 � 63.9 mm3. The lengths of all cen-terlines of all branches were added to attain the totalairway length. An illustration of the centerlines is givenin Fig. 3. The average lower airway length was 174.49 �37.96 mm. By dividing the airway volume by the airwaylength one attained an average cross sectional area(CSA) of the airways. On average this value was 1.95 �0.40 mm2. All values for rats 1–7, in which the tracheo-bronchial tree was scanned, can be found in Table 1.

As indicated in Fig. 3 the detected tracheobronchialtree in each rat is slightly different in terms of volumeand the number of airways. To investigate the variabilitybetween the models a correlation was made between theaverage CSA and the airway length of a model (Fig. 4).Rats 1–7 are shown in Fig. 4 and the CSA valuestowards the origin are those of the mean trachea valuesfor all assessed animals.

Fig. 1. Definition of scanning length for rat airway model.

722 BACKER ET AL.

An exponential correlation was found with equationwith a coefficient of variation r2 ¼ 0.9789.

AverageCSA ¼ 4:4258 e0:0042ðAirwayLengthÞ (1)

This correlation was made without the outliers asexplained in the discussion section. If the correlation wasvalid, this would imply that if one would take a seg-mented model and subsequently cut this model to reducethe segmented airway length, the resulting average CSAwould be predicted by the correlation equation. To testthis, the model of rat 3 which was the one with the larg-est volume was cut twice. It was found that each time theresulting average CSA was correctly given by the correla-tion equation. This is indicated by the crosses in Fig. 4.From this analysis and this correlation it could be con-cluded that the model that was segmented the furthestwas most suitable to represent the rats within this weightcategory. In addition, small changes in weight within thesize range 270–450 g have little impact on lung size. Moredetail can be found in the Discussion section.

Upper Airway

For the extra-thoracic airway geometries a slightly dif-ferent approach is taken. In Fig. 5 the morphology of thenares is presented for the euthanized rats using adynamic scanning method. The average volume of air inthe nares of the euthanized rats is 162.75 � 88.86 mm3

and for the anaesthetized rats, 75.93 � 12.27 mm3. Thecenterlines are less informative for this part of respiratorytract, because these centerlines would be calculated in thecomplex olfactory and sinus regions that only account fora small fraction of the air mass flow rate. Therefore sur-face area was calculated to better represent the nares.

The euthanized rats had an average surface area of743.5 � 451.58 mm2 and the anaesthetized rats, 668 �102.45 mm2. Division of the volume by the surface areagives a characteristic length of the nares. For the eutha-nized rats this characteristic length is 0.22 � 0.03 mmon average, whereas for the anaesthetized scanned ani-mals the average of this length is lower at 0.11 � 0.001mm. The detected characteristic length appears smallerin the anaesthetized animals, with a lower variability.

The ‘Average’ Model

A representative average model was selected using thecriteria described in the discussion section. The nasopha-ryngeal volume of the representative model was 373mm3, the tracheal volume was 173 mm3 and the volumeof the central airways amounted to 233 mm3. The result-ing model is shown in Fig. 6.

Measurement of Lung Lobes

A rat has four lung lobes in the right lung and one lobein the left lung (Greene, 1970) which can be distinguishedin the micro-CT images by following the fissure lines. Onelobe of the right lung is physically present in the left partof the rat’s thorax. Air is supplied to this lobe through thecharacteristically large airway which laterally crosses thethorax. Lung lobe measurements are shown in Fig. 6,where the airways are numbered and colored according to

the lobe they provide with air. Figure 7 presents the values

for the volumes and surface areas of the lung lobes with

the nomenclature as defined by Lee et al. (2008).

TABLE

1.Overview

ofsc

anned

rats,volu

mes,

length

s,and

cross

sectionalareas

Rat

Weight

(g)

Trach

eo-

bronch

ial

tree

euthanized

Nasa

lpassages

euthanized

Nasa

lpassages

anaesthetised

Trach

eobronch

ial

tree

volume

Trach

eobronch

ial

tree

length

Average

cross

sectional

area

Nasa

lpassages

volume

Nasa

lpassages

totalarea

Nasa

lpassages

char.length

lCT

lCT

Dynamic

lCT

(mm

3)

(mm)

(mm

2)

(mm

3)

(mm

2)

(mm)

1450

XX

–332

126

2.6

100

530

0.19

2450

XX

–331

155

2.1

294

1420

0.21

3320

XX

–386

232

1.7

119

486

0.24

4270

XX

–265

184

1.4

138

538

0.26

5390

X–

–398

208

1.9

––

–6

380

X–

–231

138

1.7

––

–7

430

X–

–384

179

2.1

––

–8

350

––

X–

––

87

752

0.12

9330

––

X–

––

63

560

0.11

10

340

––

X–

––

68

601

0.11

11380

––

X–

––

86

759

0.11

AIRWAY MORPHOLOGY IN SPRAGUE–DAWLEY RATS 723

DISCUSSION

Drug delivery through inhalation has become a popu-lar method the past decades to attain local therapeuticeffects. The inhalation route could also be an attractive

alternative to attain systemic effects because for thesame amount of medication direct delivery to the lungsresults in a higher biological availability compared tooral medication. However, because adequate technologies

Fig. 3. Rat tracheobronchial trees (ventral view) en illustration of centerlines.

Fig. 2. Respiratory gating in dynamic scans of rat upper airway.

724 BACKER ET AL.

have not been commonly used in the preclinical setting,few investigators have addressed the detailed depositionof drugs in the lung (Ewing et al., 2008). For this reason,developers of new inhalation therapies would benefitgreatly from having a representative, virtual rat modelof the airways. It is, however, impractical to develop aspecific model for each animal under investigation.Therefore the aim of this work was to select or constructthe average model of the rat, obtained through imagingtechniques, whereby the attributes of candidate formula-tions or delivery systems may be applied to assess depo-sition within its respiratory tract

The primary aim of the project was to analyze several ani-mals in the given weight category and subsequently selectthe average sized structures, giving an average animal. Theaverage animal should contain as much information as pos-sible on the airway anatomy and at the same time must berepresentative for the species in this category.

There are significant issues in determining the attrib-utes of the ‘average’ rat; namely whether the modelshould be based on the average volume, the averagelength or the average surface area. Other considerationsinclude the effect of image quality on the segmentationprocedure. The extent to which a model could be recon-structed from the segmented images depends mainly onthe amount of noise in the images. The noise level canbe influenced by the size of the animal because fat pads

attenuate the signal. Therefore selection on the basis av-erage volume or area of the models could result in a non-optimal model. From Fig. 4, it can be seen that two mod-els (4 and 6, indicated by the solid triangular symbols)appear to be outliers, that is, for the measured airwaylength the average CSA appears to be smaller than forthe other models. Inspecting the 3D reconstructed modelsand the micro-CT data revealed that these modelsappeared to be deflated during the scan which lasted �6hr. The rats were not intubated during the scans as thiswould disturb the geometry of the trachea and the upperairway. Therefore it appears that there is a correlationbetween segmented airway length and the average CSAfor all rats, except 4 and 6, where there was significantdeflation in the airways. The correlation shown in Fig. 4,excluding the outliers, shows that 98% of the variabilityin the data can be explained by the variation in the seg-mentation length. On the assumption that the errorsinvolved in the segmentation process were consistent andnegligible, the variability between rats, in this weightrange and detected volume of the tracheobronchial tree, isaround 2%. As a result, it is logical to select the modelthat was segmented the greatest length. Consequently,this was also the model with the largest tracheobronchialvolume and not the average lower airway volume. Impor-tant to note is that, unlike in human airways, the rodentairways do not have a dichotomic branching structure,

Fig. 4. Average cross sectional area as a function of airway length.

AIRWAY MORPHOLOGY IN SPRAGUE–DAWLEY RATS 725

but rather a central bronchus. This can have a significanteffect on lung function and particle deposition as will beinvestigated in a future study.

Having selected a representative tracheobronchial air-way model, the most suitable upper airway model mustnow be selected. As can be seen in Fig. 5 some differen-ces exist between the upper airway models attained inthe euthanized animals and the models from the anaes-thetized animals. In the so-called ‘nasal cavity’ region,indicated in Fig. 5, the differences are more apparent. Inthe dynamic scans taken while the rat was breathing,this very narrow region shows a small oscillatory dilat-ing movement. It appears to be that the airways in thisregion collapse once the animals are euthanized. This isconfirmed by the calculation of the characteristic length.For the anaesthetized animals this value is almost con-stant with a very small variability. For the euthanizedanimals this value is higher and also the variability islarger. To construct a representative model the upperairway morphology must be adapted to reflect the behav-ior as observed in the living animals. Therefore the rat

with the best defined nasal geometry was selected to berepresentative of a ‘living’ rat. By means of Booleanoperations the results of the snout and nasal passagesfrom this rat was merged with the scan of the tracheo-bronchial tree to form an average and representativemodel of the rat’s respiratory system.

CONCLUSION

Combining high resolution micro-CT scans withdynamic micro-CT scans made it possible to create a rep-resentative virtual geometry of the respiratory system ofthe Sprague–Dawley rats. It was possible to identify an-atomical landmarks and a correlation was foundbetween the segmented airway length and the averageof the airways’ CSA. The micro-CT images contained suf-ficient detail to identify the fissure lines in the lungs, en-abling reconstruction and measurement of lobularvolumes. The virtual model will now be used to simulatefluid flow conditions during respiration and hence toassess deposition patterns of inhaled particles.

Fig. 5. Rat nasal passages.

726 BACKER ET AL.

ACKNOWLEDGMENTS

The authors thank Arabe Ahmed from GlaxoSmithK-line for the assistance in data gathering. They acknowl-edge Ir. Frank Lakiere and Dr. Andrei Postnov from theUniversity of Antwerp for the technical assistance dur-ing the scanning of the animals.

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Fig. 7. Volume and surface area measurements of the lung lobesfor the representative rat model.

Fig. 6. Mean rat model (left, ventral view), lobular segmentation(top right), and airways colored by lobular pathway (bottom right).

AIRWAY MORPHOLOGY IN SPRAGUE–DAWLEY RATS 727