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Study of the Variability in Upper and Lower Airway Morphology in Sprague УDawley Rats Using Modern Micro-CT Scan-Based Segmentation Techniques.

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THE ANATOMICAL RECORD 292:720–727 (2009)
Study of the Variability in Upper and
Lower Airway Morphology in Sprague–
Dawley Rats Using Modern Micro-CT
Scan-Based Segmentation Techniques
FluidDA, Drie Eikenstraat 661, 2650 Edegem-Antwerp, Belgium
Vision Lab, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk–Antwerp, Belgium
Department of respiratory medicine, University Hospital Antwerp, Wilrijkstraat 10,
2650 Edegem, Antwerp, Belgium
GlaxoSmithKline Research and Development, Park Road, Ware, Hertfordshire,
SG12 0DP, UK
Skyscan, Kartuizersweg 3B, 2550 Kontich, Belgium
Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1,
2610 Wilrijk–Antwerp, Belgium
Animal models are being used extensively in pre-clinical and safety
assessment studies to assess the effectiveness and safety of new chemical
entities and delivery systems. Although never entirely replacing the need
for animal testing, the use of computer simulations could eventually
reduce the amount of animals needed for research purposes and refine
the data acquired from the animal studies. Computational fluid dynamics
is a powerful tool that makes it possible to simulate flow and particle
behavior in animal or patient-specific respiratory models, for purposes of
inhaled delivery. This tool requires an accurate representation of the respiratory system, respiration and dose delivery attributes. The aim of this
study is to develop a representative airway model of the Sprague–Dawley
rat using static and dynamic micro-CT scans. The entire respiratory tract
was modeled, from the snout and nares down to the central airways at
the point where no distinction could be made between intraluminal air
and the surrounding tissue. For the selection of the representative model,
variables such as upper airway movement, segmentation length, airway
volume and size are taken into account. Dynamic scans of the nostril
region were used to illustrate the characteristic morphology of this region
in anaesthetized animals. It could be concluded from this study that it
was possible to construct a highly detailed representative model of a
Sprague–Dawley rat based on imaging modalities such as micro-CT scans.
C 2009 Wiley-Liss, Inc.
Anat Rec, 292:720–727, 2009. V
Key words: small animal imaging; micro-CT; rat; respiratory
tract; 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.
Received 4 July 2008; Accepted 11 December 2008
DOI 10.1002/ar.20877
Published online 25 March 2009 in Wiley InterScience (www.
Animal models are being used extensively in pre-clinical and safety assessment studies to assess the effectiveness and safety of new chemical entities, delivery systems
or 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 the
product for a longer periods of time (Wagner et al., 2006;
Lee et al., 2007) or for shorter periods of time to assess
acute effects (Dong et al., 2005; Kouadio et al., 2005). As
in human trials (De Backer et al., 2007a) a trend is
emerging in animal studies towards accurate computer
models describing the animals’ physiological behavior
(Schroeter et al., 2006; Galle et al., 2007). Although never
entirely replacing animal studies, this could, eventually,
reduce the amount of animals needed for research.
The increasing prevalence of respiratory diseases such
as asthma and chronic obstructive pulmonary disease
(COPD) intensified the research regarding the morphology and physiology of the respiratory system both clinically as well as pre-clinically. A tool that is increasingly
used to assess the respiratory function is computational
fluid dynamics or CFD. This method is capable of simulating flow behavior in virtual models (Bush et al., 1998;
Andersen et al., 2000; Minard et al., 2006). The challenge
is to develop mathematical models that realistically
reflect the respiratory physiology of the animal by accurately simulating its fluid flow conditions. These models
comprise of accurate geometrical representations of the
airway structures and realistic boundary conditions
which drive the flow (De Backer et al., 2007b). The CFD
method and these models can assist in the development
of new inhalation therapies and the optimization of
inhaler devices (Coates et al., 2007; Kleinstreuer et al.,
2007). Advances in small animal imaging using micro-CT
or MRI have made it possible to study the respiratory
system in a specific animal into great detail (Johnson,
2007; Lam et al., 2007; Wietholt et al., 2008). Earlier
work by Chaturvedi and Lee (Chaturvedi and Lee, 2005)
has used micro CT images to accurately study the morphology of the mouse and canine airway tract based on
lung casts and segmentation principles. The study we
present here uses similar techniques but scans are performed in vivo or in situ. Our study aims to develop a
representative, realistic model of the rat respiratory tract
based on high-resolution micro-CT scans. In particular
the variability between different animals from the same
species will be investigated as also performed in another
recent study by Lee et al. (Lee et al., 2008). Lee et al.
again used lung casts to assess the geometrical features
of the respiratory system of healthy Sprague–Dawley
rats. In our study, a range of techniques (static and
dynamic micro-CT scans) will be used in different circumstances (anaesthetized and euthanized animals) to obtain
in vivo and in situ images. Differences between different
rats were assessed to obtain a representative model. Both
the nasal passages and the tracheobronchial tree were
considered in the model. The resulting representative
model could subsequently be used in flow and particle
deposition simulation studies.
In this study a total of 11 Sprague–Dawley rats were
used. 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 snout
and entire respiratory tract of the rats. The scanner had
a tube diameter of 68 mm and a 17 mm single scan
length. For the scans a resolution of 35 lm was selected.
An initial test comparing scans from anaesthetized with
euthanized rats clearly showed that resolution of airway
structures in the euthanized rat was superior to the
anaesthetized rat. Therefore, the airways in the lungs
were scanned in 7 euthanized rats to optimize the image
quality. The tracheobronchial tree was defined as the
airways starting from the larynx down. The nasal passages were scanned in a total of 8 rats. A definition of
the airway regions is given in Fig. 1 and an overview of
the scans is provided in Table 1. In addition to the volume of the lower airways, the airway length was determined through the construction of centerlines based on
the segmentation masks
lCT Scanning of the Respiratory System
The first rats (rat 1–4) were scanned from the nostrils
down to the diaphragm to include the lungs and all extrathoracic 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 were
taken with a source voltage of 100 kV and a source current
of 140 lA. The resolution was set to 35 lm and the rotation
step was 0.6 degrees. The rats needed to be euthanized for
these scans because the scanning time would have taken
too long (>5 hr) to bring animals under anesthesia and to
preserve image quality due to motion artifacts. The animals were euthanized with 1 mL of Nembutal and were
left for 1 hr to complete the state of rigor mortis. To
increase the number of animal-specific tracheobronchial
morphologies, only the tracheobronchial regions of 3 additional rats (rats 5–7) were scanned additionally.
Dynamic lCT Scanning of the Nasal Passages
Rats are obligate nasal breathers, using their sense of
smell to identify and classify objects. Consequently, the
nasal passages are well developed and have an important filtering function to prevent hazardous particles
from entering the animals’ respiratory system. To assess
the movement of the nostril and upper airway region
during breathing, dynamic scans of this region were
taken in 4 rats (rats 8–11) in addition to the static lCT
scans of rats 1–4. Scans were taken with a source voltage of 70 kV and a source current of 140 lA. The resolution was set to 35 lm and the rotation step was 0.9
degrees. The scan length was 1.5 cm. The rats were
anaesthetized and their breathing pattern was monitored. The dynamic scans were performed using respiratory gating. The animals were anaesthetized with a 5050 mixture of Nembutal and saline. The dosage was
based on 35 mg of Nembutal per 2.5 kg of bodyweight.
The respiratory gating was based on a visual triggering
system. The motion of the animal’s thorax was gated
and whenever a clear signal was detected four images
were taken and the registration time was recorded (Fig.
2). Afterwards all images were sorted into several bins
to attain static images at certain breathing levels.
Therefore images of the entire region of interest where
obtained for the normal inspiration and expiration.
Fig. 1. Definition of scanning length for rat airway model.
Combining these static images produced a dynamic
image, indicating the changes over time.
Airway Segmentation
All scans from both anaesthetized and euthanized animals were then read into a commercially available and
validated software package Mimics (Materialise, Leuven,
Belgium). Three-dimensional reconstructions of the
upper and lower airways were made, based on segmentation principles. Voxels with Hounsfield Units, a measure of electron density, within the pre-described range
(1,024 to 865) were placed in a separate mask. A
three-dimensional virtual representation of the structure
was reconstructed from this mask. Airway lengths, surfaces, and volumes could be measured from this reconstruction. The selection process for the ‘average animal
model’ is explained in the results and discussion section.
Segmentation into the various lung lobes was carried
out using the ‘average model’. This was done by segmenting the lung volumes and subsequently defining the
fissures separating the lung lobes.
Ethical Approval
This study has been approved by the ethical committee of the University of Antwerp. Animals were handled
by the qualified staff of the animalarium of the University of Antwerp.
Lower Airway
A visual representation of the reconstructed lower airways of rat 1–7 can be found in Fig. 3. The typical structure of the airway tree can be clearly seen in Fig. 3.
Measurements of the segmented lower airway volume
gave an arithmetic mean airway volume and standard
deviation of 332.40 63.9 mm3. The lengths of all centerlines of all branches were added to attain the total
airway length. An illustration of the centerlines is given
in Fig. 3. The average lower airway length was 174.49 37.96 mm. By dividing the airway volume by the airway
length 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 tracheobronchial tree was scanned, can be found in Table 1.
As indicated in Fig. 3 the detected tracheobronchial
tree in each rat is slightly different in terms of volume
and the number of airways. To investigate the variability
between the models a correlation was made between the
average CSA and the airway length of a model (Fig. 4).
Rats 1–7 are shown in Fig. 4 and the CSA values
towards the origin are those of the mean trachea values
for all assessed animals.
An exponential correlation was found with equation
with a coefficient of variation r2 ¼ 0.9789.
Average CSA ¼ 4:4258 e0:0042ðAirway LengthÞ
This correlation was made without the outliers as
explained in the discussion section. If the correlation was
valid, this would imply that if one would take a segmented model and subsequently cut this model to reduce
the segmented airway length, the resulting average CSA
would be predicted by the correlation equation. To test
this, the model of rat 3 which was the one with the largest volume was cut twice. It was found that each time the
resulting average CSA was correctly given by the correlation equation. This is indicated by the crosses in Fig. 4.
From this analysis and this correlation it could be concluded that the model that was segmented the furthest
was most suitable to represent the rats within this weight
category. In addition, small changes in weight within the
size range 270–450 g have little impact on lung size. More
detail can be found in the Discussion section.
Dynamic lCT
For the extra-thoracic airway geometries a slightly different approach is taken. In Fig. 5 the morphology of the
nares is presented for the euthanized rats using a
dynamic scanning method. The average volume of air in
the nares of the euthanized rats is 162.75 88.86 mm3
and for the anaesthetized rats, 75.93 12.27 mm3. The
centerlines are less informative for this part of respiratory
tract, because these centerlines would be calculated in the
complex olfactory and sinus regions that only account for
a small fraction of the air mass flow rate. Therefore surface area was calculated to better represent the nares.
The euthanized rats had an average surface area of
743.5 451.58 mm2 and the anaesthetized rats, 668 102.45 mm2. Division of the volume by the surface area
gives a characteristic length of the nares. For the euthanized rats this characteristic length is 0.22 0.03 mm
on average, whereas for the anaesthetized scanned animals the average of this length is lower at 0.11 0.001
mm. The detected characteristic length appears smaller
in the anaesthetized animals, with a lower variability.
The ‘Average’ Model
tree volume
A representative average model was selected using the
criteria described in the discussion section. The nasopharyngeal volume of the representative model was 373
mm3, the tracheal volume was 173 mm3 and the volume
of the central airways amounted to 233 mm3. The resulting model is shown in Fig. 6.
Measurement of Lung Lobes
tree length
Upper Airway
TABLE 1. Overview of scanned rats, volumes, lengths, and cross sectional areas
total area
char. length
A rat has four lung lobes in the right lung and one lobe
in the left lung (Greene, 1970) which can be distinguished
in the micro-CT images by following the fissure lines. One
lobe of the right lung is physically present in the left part
of the rat’s thorax. Air is supplied to this lobe through the
characteristically large airway which laterally crosses the
thorax. 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).
Fig. 2. Respiratory gating in dynamic scans of rat upper airway.
Fig. 3. Rat tracheobronchial trees (ventral view) en illustration of centerlines.
Drug delivery through inhalation has become a popular method the past decades to attain local therapeutic
effects. The inhalation route could also be an attractive
alternative to attain systemic effects because for the
same amount of medication direct delivery to the lungs
results in a higher biological availability compared to
oral medication. However, because adequate technologies
Fig. 4. Average cross sectional area as a function of airway length.
have not been commonly used in the preclinical setting,
few investigators have addressed the detailed deposition
of drugs in the lung (Ewing et al., 2008). For this reason,
developers of new inhalation therapies would benefit
greatly from having a representative, virtual rat model
of the airways. It is, however, impractical to develop a
specific model for each animal under investigation.
Therefore the aim of this work was to select or construct
the average model of the rat, obtained through imaging
techniques, whereby the attributes of candidate formulations or delivery systems may be applied to assess deposition within its respiratory tract
The primary aim of the project was to analyze several animals in the given weight category and subsequently select
the average sized structures, giving an average animal. The
average animal should contain as much information as possible on the airway anatomy and at the same time must be
representative for the species in this category.
There are significant issues in determining the attributes of the ‘average’ rat; namely whether the model
should be based on the average volume, the average
length or the average surface area. Other considerations
include the effect of image quality on the segmentation
procedure. The extent to which a model could be reconstructed from the segmented images depends mainly on
the amount of noise in the images. The noise level can
be influenced by the size of the animal because fat pads
attenuate the signal. Therefore selection on the basis average volume or area of the models could result in a nonoptimal model. From Fig. 4, it can be seen that two models (4 and 6, indicated by the solid triangular symbols)
appear to be outliers, that is, for the measured airway
length the average CSA appears to be smaller than for
the other models. Inspecting the 3D reconstructed models
and the micro-CT data revealed that these models
appeared to be deflated during the scan which lasted 6
hr. The rats were not intubated during the scans as this
would disturb the geometry of the trachea and the upper
airway. Therefore it appears that there is a correlation
between segmented airway length and the average CSA
for all rats, except 4 and 6, where there was significant
deflation in the airways. The correlation shown in Fig. 4,
excluding the outliers, shows that 98% of the variability
in the data can be explained by the variation in the segmentation length. On the assumption that the errors
involved in the segmentation process were consistent and
negligible, the variability between rats, in this weight
range and detected volume of the tracheobronchial tree, is
around 2%. As a result, it is logical to select the model
that was segmented the greatest length. Consequently,
this was also the model with the largest tracheobronchial
volume and not the average lower airway volume. Important to note is that, unlike in human airways, the rodent
airways do not have a dichotomic branching structure,
Fig. 5. Rat nasal passages.
but rather a central bronchus. This can have a significant
effect on lung function and particle deposition as will be
investigated in a future study.
Having selected a representative tracheobronchial airway model, the most suitable upper airway model must
now be selected. As can be seen in Fig. 5 some differences exist between the upper airway models attained in
the euthanized animals and the models from the anaesthetized animals. In the so-called ‘nasal cavity’ region,
indicated in Fig. 5, the differences are more apparent. In
the dynamic scans taken while the rat was breathing,
this very narrow region shows a small oscillatory dilating movement. It appears to be that the airways in this
region collapse once the animals are euthanized. This is
confirmed by the calculation of the characteristic length.
For the anaesthetized animals this value is almost constant with a very small variability. For the euthanized
animals this value is higher and also the variability is
larger. To construct a representative model the upper
airway morphology must be adapted to reflect the behavior as observed in the living animals. Therefore the rat
with the best defined nasal geometry was selected to be
representative of a ‘living’ rat. By means of Boolean
operations the results of the snout and nasal passages
from this rat was merged with the scan of the tracheobronchial tree to form an average and representative
model of the rat’s respiratory system.
Combining high resolution micro-CT scans with
dynamic micro-CT scans made it possible to create a representative virtual geometry of the respiratory system of
the Sprague–Dawley rats. It was possible to identify anatomical landmarks and a correlation was found
between the segmented airway length and the average
of the airways’ CSA. The micro-CT images contained sufficient detail to identify the fissure lines in the lungs, enabling reconstruction and measurement of lobular
volumes. The virtual model will now be used to simulate
fluid flow conditions during respiration and hence to
assess deposition patterns of inhaled particles.
Fig. 6. Mean rat model (left, ventral view), lobular segmentation
(top right), and airways colored by lobular pathway (bottom right).
Fig. 7. Volume and surface area measurements of the lung lobes
for the representative rat model.
The authors thank Arabe Ahmed from GlaxoSmithKline for the assistance in data gathering. They acknowledge Ir. Frank Lakiere and Dr. Andrei Postnov from the
University of Antwerp for the technical assistance during the scanning of the animals.
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