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Application of micro-CT in small animal imaging

Sebastian J. Schambach a, Simona Bag a, Lothar Schilling b, Christoph Groden a, Marc A. Brockmann a,*

a University of Heidelberg, Medical Faculty Mannheim, Department of Neuroradiology, Germanyb University of Heidelberg, Medical Faculty Mannheim, Division of Neurosurgical Research, Germany

a r t i c l e i n f o

 Article history:

Accepted 21 August 2009

Available online 23 August 2009

Keywords:

Micro-computed tomography (lCT)

Imaging

Preclinical studies

Rodent

Mouse

Rats

Mice

Small animal

In vivo studies

a b s t r a c t

Over the past decade, the number of publications using micro-computed tomography (lCT) imaging in

preclinical in vivo studies has risen exponentially. Higher spatial and temporal resolution are the keytechnical advancements that have allowed researchers to capture increasingly detailed anatomical

images of small animals and to monitor the progression of disease in small animal models. The purpose

of this review is to present the technical aspects of  lCT, as well as current research applications. Our

objectives are threefold: to familiarize the reader with the basics of  lCT techniques; to present the type

of experimental designs currently used; and to highlight limitations, future directions, in  lCT-scanner

research applications, and experimental methods. As a first step we present different  lCT setups and

components, as well as image contrast generation principles. We then present experimental approaches

in order of the evaluated organ system. Finally, we provide a short summary of some of the technical

limitations of  lCT imaging and discuss potential future developments in  lCT-scanner techniques and

experimental setups.

 2009 Published by Elsevier Inc.

1. Introduction

Small animals are essential as models of human disease and the

study of organism development. Small animal imaging has a vital

role in understanding these models and a key role in phenotyping,

as well as drug development and treatment. In the early 1970s,

clinical imaging was revolutionized by the introduction of com-

puted tomography (CT). Until then, the examination of small

rodents in research projects, especially of mice and rats, was

limited by the relatively low geometrical resolving capacity of 

clinical CT scanners to   1 mm3 [1]. Over the past three decades,

micro-CT (lCT) imaging has rapidly advanced with higher quality

resolution, the introduction of the cone beam reconstruction

algorithm, and an increased availability of dedicated scanners for

non-invasive small animal imaging research   [2]. This increased

use of  lCT has been reflected in a rising number of publicationsbeginning in the early 1980s. Fig. 1 graphically depicts this rising

number of annual publications of   lCT in preclinical research,

underlining the increased importance of these scanners. This graph

is based on a simple query of the public database PubMed using

the mesh terms:   lCT or MICRO-CT or ‘‘High Resolution CT” or

Mini-CT and ANIMAL.

Initially lCT demonstrated excellent spatial resolution, but poor

soft tissue contrast. Therefore, early publications implementing

lCT mainly focused on the non-invasive evaluation of high con-

trast structures, such as bones or implants. With advancements

in X-ray detector sensitivity, notable improvements were made

both in temporal and in geometrical resolution, as well as readout

speed. In addition, with the introduction of new contrast agents to

elevate soft tissue contrast,   lCT could be transferred to in vivo

applications in preclinical research to evaluate soft tissue

structures and vessel morphology.

The primary purpose of this review is to familiarize the reader

with the underlying technical aspects and application possibilities

of  lCT imaging in experimental small animal imaging. The objec-

tives of this review are threefold: first, to present the technical fun-

damentals of    lCT; second, to describe successfully applied

experimental   lCT setups including various contrast generationmechanisms and contrast enhancement possibilities in relation

to the examined organ system; and finally, to identify current

limitations of  lCT-imaging and future directions.

2. Technical aspects of  lCT imaging 

Since the first description of  lCT use in preclinical research in

the early 1980s [3–6], a number of reviews of the technology and

applications of lCT have been published [1,7–15]. Initially, numer-

ous small companies specialized in the production of dedicated

small animal   lCT scanners, but they were subsequently bought

by larger competitors with growing interests in  lCT technology.

1046-2023/$ - see front matter  2009 Published by Elsevier Inc.doi:10.1016/j.ymeth.2009.08.007

*   Corresponding author. Address: University of Heidelberg, Medical Faculty

Mannheim, Department of Neuroradiology, Theodor-Kutzer-Ufer 1-3, 68167

Mannheim, Germany. Fax: +49 621 383 2165.

E-mail address: brockmann@gmx.de (M.A. Brockmann).

Methods 50 (2010) 2–13

Contents lists available at  ScienceDirect

Methods

j o u r n a l h o m e p a g e :   w w w . e l s e v i e r . c o m / l o c a t e / y m e t h

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General Electric acquired Enhanced Vision System Corp. (EVS)

in the year 2002; Siemens bought ImTek Inc. in 2004 and CTI

Molecular Imaging Inc. in 2005, while Varian Security & Inspection

Products acquired Bio-Imaging Research Inc. (BIR) in 2007. A short

market overview can be found in Table 1. To the best of our knowl-

edge, this is a comprehensive overview of the market at the time of 

this review. We recognize, however, that other suppliers may also

exist of which we have not listed here.

Fig. 1.  The rising number of annual publications of  lCT in preclinical research demonstrates the increasing importance of these scanners. This graph is based on a simple

query of the public database PubMed with using the following mesh terms: lCT or MICRO-CT or ‘‘High Resolution CT” or Mini-CT and ANIMAL. A time line was created with

MEDSUM: an online MEDLINE summary tool by Galsworthy, MJ. Hosted by the Institute of Biomedical Informatics (IBMI), Faculty of Medicine, University of Ljubljana,

Slovenia. URL: www.medsum.info.

 Table 1

Market overview on micro-CT: manufacturers and their current products.

Company Web site Products

Biomedical Imaging Research (BIR)   http://www.bio-imaging.com   ACTIS 150/90 Desktop

Varian Inc. ACTIS 150/130 Desktop

Lincolnshire, IL, USA ACTIS 200/225 Desktop

Bioscan, Inc.

4590 MacArthur Blvd., NW, USAWashington, DC 20007

http://www.bioscan.com   NanoSPECT/CT

NanoPET/CT

Echo Medical Systems

Houston, TX, USA

http://www.echomri.com   LaTheta LCT-200

LaTheta LCT-100A

Gamma Medica-ldeas, Inc.

Northridge, CA, USA

http://www.gm-ideas.com   FLEX Triumph

GE Medical Systems

Waukesha, Wl, USA

http://www.gehealthcare.com   eXplore Locus

eXplore Locus SP

eXplore CT 120

eXplore Vista PET/CT

Triumph

SCANCO Medical AG

Brüttisellen, Switzerland

http://www.scanco.ch   viva CT 75

viva CT 40

extremCT

lCT 35

lCT 40

lCT 80

Siemens AG

Erlangen, Germany

http://www.medical.siemens.com   Inveon Micro CT

SkyScan

Kontich, Belgium

http://www.skyscan.be   SkyScan 1076 in-vivo

SkyScan 1178 high-throughput

SkyScan 1172 high-resolution

SkyScan 1174 compact

Stratec Medizintechnik GmbH

Pforzheim, Germany

http://www.stratec-med.com   XCT Research SA/SA+

XCT 3000 Research M/M+

Orthometrix Inc.

Naples, FL, USA

http://www.orthometrix.net   XCT FAN Beam l-Scope

VAMP GmbH

Erlangen, Germany

http://www.vamp-gmbh.de   TomoScope 30s

TomoScope 30s+

TomoScope Duo

YXLON International GmbH

Garbsen, Germany

http://www.yxlon.com   Yxlon Y.Fox  lCT

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In the literature, both customized non-destructive testing  lCTs

[16]  and custom-made  lCTs  [3,17]  adapted for the necessities of animal research are described. The following paragraphs will detail

some of the various  lCT scanner designs and discuss the advanta-

ges and limitations of this form of imaging technology.

 2.1.  lCT setup

 2.1.1. Construction principles

Generally, there are two different construction principles with

respect to  lCT scanners. The first construction principle involves

scanners with an X-ray detector and radiation source mounted

on a gantry that is rotated around the examined object (Fig. 2A).

In these scanners the source–detector distance (SDD) is construc-

tion-conditioned in most marketed tomographs (with a few excep-

tion where SDD can be adjusted), with a magnification level for theobject in the course of the beam. In these systems the achievable

geometrical resolution depends mainly on the pixel pitch and

matrix size of the detector used, as well as the focusing mode of 

the X-ray tube used. Accordingly, scanners are named ‘‘mini-CT”

because the setup is analogous to clinical CTs.

The second construction principle, which is found mostly in

ex vivo specimen  lCT scanners and custom built systems, is out-

lined in Fig. 2B. In this design the examined object is rotated within

the light path. The setup allows the free adjustment of source–

object distance (SOD) and object–detector distance (ODD), thereby

allowing SDD adjustment. Free adjustment of SOD and ODD facili-

tates optimization of the geometric magnification level, depending

on the signal-to-noise ratio (SNR) and the penumbra blurring [15].

Thus, for small fields of view, higher maximal resolution can be

achieved as compared to a conventional setup. In these systems,

the object can be rotated horizontally   [16]   or vertically   [17]

orthogonal to the ray path. However, one drawback is the necessity

to fix the examined animal during rotation around its own axis, to

prevent movement blurring in the resulting datasets.

 2.1.2. X-ray tubes

The availability of a variety of   lCT scanners using a range of 

X-ray tube technologies presents both advantages and disadvan-

tages. The majority of marketed scanners use nano- or microfocus

X-ray tubes with transmission targets. In these X-ray tubes an

electron beam is produced on the tip of a hairpin tungsten filament

and focused by several magnetic lenses onto a focal spot of 

1–10 lm on a transmission target (Fig. 3).

Transmission targets typically have a thin layer of tungsten

(about 100 lm) electroplated or vapor-deposited on a carrying

material with low atomic number and high thermal conductance

such as beryllium or chemical vapor depositioned (CVD)-diamonds(e.g. Diamond Materials GmbH, Freiburg, Germany). On the outside

of the transmission targets a cone-shaped beam of braking radia-

tion (depending on tube voltage), and characteristic radiation

(depending on the target material) is produced which irradiates

onto a digital X-ray detector.

In contrast to this, in conventional clinical CT or three-dimen-

sional (3D)-rotation angiography systems, X-ray tubes with reflec-

tion targets and focal spot sizes of a minimum of 300lm are

typically used. If tubes of this dimension are used, as per the pro-

tocol of Badea et al. [17], then a low magnification must be used in

order to minimize penumbra blurring caused by the large focal

spot. Reflection target X-ray tubes convert the irradiated electron

energy less efficiently to X-ray photons than transmission target

tubes [18]. On the other hand, reflection targets can absorb moreheat energy without damage because they have a thicker tungsten

anode compared to the tungsten layer on transmission targets.

Therefore, higher energy electron beams can be used in reflection

anode tubes, leading to higher photon flux in these tubes  [17].

Reflection anode tubes typically generate power in the range of 

kilowatts, whereas microfocus transmission tubes operate in the

range of watts. In 1994, Flynn et al. stated that the output power

of a microfocus tube would generally follow   P max = 1.4( x)0.88

[W/lm] where x  equals the focal spot size in  lm [19]. Due to the

use of advanced target materials, such as CVD-diamonds that have

Fig. 2.   Different lCT architectures. In thedesign illustrated under (A), theexamined

object is placed still in the center of the setup and a gantry carrying detector and X-

ray source is rotated around it. The geometrical magnification factor is fixed

structurally by the defined SDD. In the setup outlined in (B), the object is rotated in

the course of the beam and can be freely positioned between detector and source,

which allows for the adjustment of the magnification level.

Fig. 3.  (A) Transmission target X-ray tube of the Y.Fox  lCT. (B) Sketch of the inside configuration of transmission tubes with the electron beam exiting a hairpin filament

(triangle) that is focused viamagneticlenses(gray bars) on thetransmission target(1) . (C) Reflection anode X-ray tube with rotating anode in closed design and(D) sketchof 

a reflecting anode X-ray tube design with electrons exiting from a curled heating cathode and the electrons accelerated onto a reflection target (1). (Image source of (C) and(D): Wikipedia Commons.)

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an extremely high warmth conductance, current microfocus tubes

far exceed these conditions. For example, the Y.Fox lCT can gener-

ate up to 13 W of power with a focal spot size of 1 lm and is lim-

ited only by the maximal power output of the voltage generator,

where a maximal output of 1.34 W would be calculated for the cor-

responding focal spot size.

 2.1.3. Beam geometry

Differences between system types also exist in terms of beam

geometry. Using a fan beam X-ray source a 3D dataset is acquired

either plane by plane (incrementally) or under continuous feed

motion of source against object (spiral-CT) [20] via a line detector.

An alternative to fan beam CT is the so-called cone beam CT that

was first implemented by Feldkamp et al.   [46,122]   (also known

as volume-CT). These principles are outlined schematically in

Fig. 4. In cone beam  lCT small objects are captured completely

in one rotation which considerably speeds up imaging compared

to incremental or spiral scanning. Cone beam  lCT also facilitates

cardiac and general thoracic imaging in small rodents with faster

physiological heart and breathing movements. Relinquishing therotation of the object, these systems also have the capability to

perform 2D measurements such as conventional X-ray, or digital

subtraction angiography (DSA) due to the large field of view cover-

age by the cone-shaped beam (Figs. 4B and 6).

 2.1.4. X-ray detectors

To record the desired image data in commercial  lCT systems,

most often charge-coupled device (CCD) photodetectors coupled

to a scintillator by tapered glass fibers are used  [21]. These detec-

tors do not have any readout electronics covering the photosensi-

tive layer, resulting in a so-called fill factor of 100% and leading to a

high signal yield. However, readout is facilitated by shift register

readout with one signal output over the entire detector, leading

to longer readout times.The closest competitors are active matrix flat panel imagers

(AMFPI) that consist of an array of photodiodes connected by a ma-

trix of thin film transistors (TFTs) such as TFT liquid crystal image

displays. The pixels are read out row by row speeding up readout

times compared to CCD detectors  [22], but the TFTs cover up to

50% of the photosensitive layers (fill factor6 50%) and reduce light

sensitivity.

 2.1.5. Contrast generation and enhancement possibilities

Contrast in  lCT images is produced most of the time by atten-

uation of X-rays in the examined sample [23]. Below 25 keV, X-ray

attenuation mainly takes place by the photoelectric effect, and the

resulting X-ray energy after attenuation is anti-proportional to the

third power of the atomic number. At higher X-ray energy levelsCompton scattering is the main cause of attenuation leading to

an anti-proportional dependency of attenuated X-ray energy to

the atomic number divided by 2. Accordingly, at low X-ray energy

the contrast between different tissues containing a mixture of ele-

ments with different atomic numbers is much higher than the

threshold of 25 keV [24].

X-ray contrast can be increased by various physical methods

such as K-edge subtraction   [25–28], X-ray phase delay contrast

[29–32]   and X-ray scatter contrast  [33–36]. With these methods,

up to 15 lg iodine/cm3 [37], 250 lg iodine/cm3, and 6 mg iodine/

cm3 [38]   can be detected, respectively, compared to 10 mg

iodine/cm3 without using the above-mentioned physical methods

for contrast enhancement.

lCT offers good image quality for objects consisting of elements

with high atomic number such as bones, but possesses relatively

weak soft tissue contrast. Therefore when imaging soft tissues,

the administration of a contrast agent frequently is desirable and

sometimes indispensable.

With regard to circulation time, two different types of contrast

agents are generally available for in vivo imaging: (a) a conven-

tional iodinated contrast medium that is eliminated from the blood

immediately by the kidneys or (b) a blood-pool contrast agent,

with a higher blood-pool half-life.

In 2004 Ritman [13] noted that up until then it was not possible

to image a bolus of an injected contrast agent within a live rodent

even with fast synchrotron scanning lCTs. Since then, faster X-ray

detectors have been developed to read out between 30 and 60 fps

and allow for angiography using conventional contrast agents.

Water-soluble, non-ionic iodine-based contrast agents are gener-

ally used in humans and are eliminated immediately from the

blood after intravenous injection. However, in humans, it is also

possible to acquire images in the first pass of the contrast agent,

due to a relatively long circulation time, as compared to the rodent,

and a sufficient temporal resolution found in clinical CTs. The rapid

elimination of non-ionic iodine-based contrast agents from the

blood restricts the administration of this contrast agent to a small

percentage of the total human blood volume. Due to very short cir-

culation times in rodents [39] a first-pass effect cannot be used asin humans. The contrast agent is eliminated from the blood within

seconds, leading to a need for continuous injection of a contrast

agent above the renal elimination rate, and throughout the scan-

ning time [40]. Previous studies have demonstrated sufficient ves-

sel contrast with an injection volume of 350 ll/30 g mouse, which

was hemodynamically well tolerated by all animals [16]. However,

a reduction of injection volume is desirable to minimize the

physiological alterations in the model organisms by intravenous

volume burden.

Experimental setups with longer scan times can benefit from

blood-pool contrast agents, consisting of molecules that bind to

plasma proteins  [41]  or macromolecules that are eliminated at a

slower rate because of the sheer size of these molecules   [42].

Blood-pool contrast agents allow scanning times that last up toseveral hours after a single administration, whereas the level of 

contrast is generally lower than that achieved by conventional con-

trast agents. Lower levels of contrast limit the use of blood-pool

contrast agents to larger vascular structures, but allow repeated

measurement of the same animal and evaluation of eventual vessel

changes without the need for repeated contrast agent administra-

tion and volume burden. The potential for longer scanning times

helps maximize the SNR, which is dependent on the inverse loga-

rithm of the reconstruction input image number. Longer scanning

times produce sharper datasets at the cost of a high radiation dose

for the examined animal.

Newer liposome-based blood-pool contrast agents with a high-

er iodine concentration of greater than 100 mg/ml and a longer

blood-pool half-life offer improved detail perceptibility particu-larly in small anatomical structures [43].

Fig. 4.   Principles of fan beam (A) and cone beam (B)   lCT: (1) X-ray source, (2)

beam, (3) rotatedobject in the course of beam in longitudinal motion (A) or in fixed

 z -position (B), and (4) line (A) or area (B) detector.

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Another novel liposome-based contrast agent tested by Montet

et al., with an iodine content of 70 mg/ml, allows for the measure-

ment of hepatic metastasis of around 250 lm in size, as well as

delineation of the liver and spleen. Furthermore, the iodinated

liposomes have been found to be a suitable contrast agent for

vascular structures   [44]. More recently, multimodal contrast

agents are being tested and gaining considerable attention as they

contain both iodine and gadolinium and can therefore be used

either in CT or in MRI   [15,45].

3. Application of  lCT in experimental imaging 

 3.1. Osseous structures

The first advances in   lCT technique were mainly driven by

imaging needs for the evaluation of bone anatomy and density

[46,47]. These publications have investigated bone density  [48];

osteogenesis   [49]; ovariectomy   [50]  and osteoporosis  [51]; bone

resorption   [52]; bone remodeling  [53]; bone regeneration  [54]

and fracture healing [55]; bone neoplasm [56] and biocompatible

materials   [57]; and many more topics. Various reviews have also

addressed the use of lCT in the evaluation of pathological changesin bone structure  [48,58–60].

The high X-ray density of osseous structures allows the precise

lCT-based evaluation of stereology, volume, and trabecular archi-

tecture of bones at micrometer resolution  [61]. For the volumetric

estimation of bone density, ex vivo lCT is described as the method

of choice [62]. Furthermore, the non-invasive quality of lCT allows

for the observation of bone structure before and after exposure to

mechanical stress under experimental conditions [12]. An isotropic

resolution of about 50 lm is described as sufficient to evaluate

changes correlated with osteoarthritis [63] and other bone-remod-

eling processes in rats in vivo  [64]. In studies of osseous disease,

differences in trabecular structure and mineralization density,

which can have an impact on experimental setup and data collec-

tion, were compared between different mouse strains in vivo  [61].

The results showed a higher degree of mineralization in C3H/HeH

mice compared to C57BL/6 mice at identical body weight and body

size in concordance with earlier ex vivo studies  [65]. According to

this study,  lCT is capable of detecting the impact of illnesses and

therapeutic interventions on bone density and structure   [61]. To

provide an example for imaging of osseous structures, Fig. 5 shows

a murine proximal femur volume rendering of a dataset acquired

via   lCT ex vivo, where we compared a fast (A: scan time 40 s)

and a slow scan mode (B: scan time 20 min).

Next to the evaluation of changes in trabecular structures, the

quantification of osteopathologic processes is also very important.

For this purpose,  lCT is used in fundamental research in oncology

for the quantification of bone metastases  [66]. Besides osteolytic

alterations or processes, an increase in bone density, such as

osteopetrosis, manifested in op/op knock out mice, can also be

assessed using  lCT [67].

 3.2. Vascular structures

To date, approximately 60 articles have been published on the

evaluation of vasculature in small animals via   lCT. Starting in

the late 1990s, topics such as angiogenesis   [40,68,69] and neovas-

cularization  [70] were studied. In addition, the vasculature of mis-

cellaneous organ systems, both in healthy and in diseased

conditions, have been evaluated in terms of renal vasculature

[71–74]; hepatic vasculature   [75]   and portal hypertension   [76];

Fig. 5.  Volume rendering of a murine femur dataset, acquired ex vivo with a continuous scan mode lasting 40 s (A) and with an incremental scan mode having 20 minacquisition time (B) using a volume-CT.

Fig. 6.   Digital subtraction angiography of the cerebral arteries after superselective

catheterization of the common carotid artery in a rat (cranio-caudal view).

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cerebral vasculature   [16,77]; coronary arteries  [78]; and ocular

vasculature [79].

The in vivo evaluation of vascular structures in small animals

via  lCT is only possible by means of contrast agent administra-

tion, which is similar to CT angiography (CTA) in humans. The

earlier, relatively slow  lCT scanners with scanning times lasting

up to hours allowed for vessel analysis in rodents after sacrifice

of the animal   [13] using perfusion with radiopaque polymerizing

substances [77,80] or shock freezing the contrast agent perfused

specimen [81]. In ex vivo studies there is no need for X-ray dose

reduction, and no anesthesia-dependent limitations in scanning

time, therefore SNR can be maximized by long scans with high

photon flux, resulting in submicron resolution   [80]. Recent ad-

vances in X-ray detector technology, leading to faster readout

times, lager pixel matrices, higher X-ray sensitivity, and higher

SNR, allow examination times of less than a minute   [16,40].

These advances have in turn lead to a reduction of movement

artifacts, anesthesia incidents, applied radiation dose, and the

use of conventional contrast agents with   lCT angiography. Fur-

thermore, not only does using systems with cone-beam geome-

try allow computed tomography to be performed, but also

digital subtraction angiography is possible as demonstrated in

Fig. 6.

Actual examples for high-resolution 3D vessel imaging in vivo

using conventional contrast agents were reported by Kiessling

et al. and Schambach et al., when they imaged tumor supplying

vessels at resolutions of 50 lm [40] and cerebrovascular structures

in mice at 16 lm resolution, respectively [16]. Figs. 7 and 8 show

in vivo datasets of intracranial and extracranial vessels of a mouse

using a conventional contrast agent (Iomerone 300, Bracco Altana)

with 40 s scanning time. These datasets allow not only the analysis

of anatomical differences in brain vasculature between mouse

strains, but also the evaluation of acute vessel diameter alterations

between hypoxia and normoxia.

The use of so-called blood-pool contrast agents for imaging of 

vascular structures as well as parenchymal organs in small animals

is well described in the literature   [82–91]. Various thoracic and

abdominal murine vascular structures acquired via   lCT in vivo

are displayed in Fig. 9 using the liposomal contrast agent Fenestra

VC.

However, some vascular pathologies can be detected without

the application of a contrast agent. For example, Persy et al. evalu-

ated the degree of aortic calcification in a model of chronic renal

failure of rats in vivo without contrast agents anddrew conclusions

about the development and the degree of renal failure in these

animals [92].

Fig. 7.   Maximum intensity projections (MIP, A–C) and volume rendering (D) of murine cerebral vessel datasets, acquired with a  lCT in vivo using a conventional contrast

agent and bolus technique. Image (A) shows the passage of the internal cerebral artery (ICA) through the skull base and the circle of Willis incorporating the middle cerebral

artery (MCA) and the anterior cerebral artery (ACA), presented in a curved-MIP. In image (B) a transversal view of the circle of Willis of a BALB/c mouse with prominent

posterior communicating artery (PcomA) between posterior cerebral artery (PCA) and superior cerebellar artery (SCA) is shown. Image (C) represents a sagittal view of 

murine brain vessels with small branches of the azygos of the pericalosal artery (azPA) visible. The azPA is supplied in mice by a unification of both ACA called azygos of the

ACA(azACA). (D) shows a volumerendering of external cranial vessels of a mouse with arteries such as the commoncarotid a. (CCA), external carotid a. (ECA),internal carotida. (ICA), stapedial a. (SA), occipital a. (OA), caudal auricular a. (CAA), superficial temporal a. (STA), facial a. (FA), and lingual a. (LA) visible.

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 3.3. Cardiothoracic imaging 

 3.3.1. Gating strategies

In this section, for practical reasons gating strategies are dis-

cussed in terms of how they are applied in cardiothoracic imaging,

rather than in a separate paragraph in Section  2.

High resolution cardiothoracic imaging of rats and mice poses a

challenge to the researcher because of the relatively small size of 

the animals and because of the high respiratory and cardiac

frequencies. The heart rate of mice ranges between 400 and 600

beats per minute (bpm), whereas the heart rate of rats is a little

bit lower at 250–400 bpm. At rest, the breathing frequency of mice

is about 200 comparedto about 70–110 respiratory cycles per min-

ute for rats. Depending on the depth and kind of anesthesia, the

heart and breathing rate can be lowered. The ultimate strategy to

control breathing during experimentation requires intubation

and controlled ventilation of laboratory animals as per the protocol

described by Namati et al. [93].

Cardiopulmonary imaging in rodents using  lCT is challenging

due to physiological motion of the cardiac and respiratory systems.

To achieve high resolution imaging of lung structures, while avoid-

ing intubation, or for cardiac imaging in vivo, the application of 

gating strategies is necessary. The blurring that is caused by the

physiological movement of the diaphragm or the heart can be

effectively reduced with cardio-respiratory gating, which can be

done either prospectively or retrospectively. In this correlation

prospective strategies are to be distinguished from retrospective

approaches, in that the latter can be subdivided into extrinsic

and intrinsic gating methods.

In prospective gating, image acquisition takes place only during

defined breathing or heart phases and requires direct synchroniza-

tion between lCT and physiologic parameters (ECG and/or breath-

ing signal) of the animal. Prospective gating is already

implemented in the majority of   lCT systems by Bioscan, GMI,

GE, Siemens, SkyScan and VAMP. An example for the successful

adoption of prospective gating is described in a publication by

Bartling et al., in which a significant improvement of image quality

was attained via prospective gating in  lCT scans of rats, rabbits

and mice [94].

Retrospective gating can be subdivided into extrinsic and intrin-

sic gating methods. Extrinsic retrospective gating requires syn-

chronization of physiological data acquired during the scanning

(e.g. ECG or respiratory movements) and the imaging data. Theassignment of the individual image projections to respiration or

heart phase is done after data acquisition [87]. However, to assign

time-points to recorded image data and physiological data for syn-

chronization requires direct hardware access to both the X-ray

detectors used and frame grabber.

Intrinsic retrospective gating can be implemented without

hardware modifications on any  lCT scanner, as long as the physi-

ological signals of heartbeat and diaphragm movement can be

sampled from the image projections itself. Of note, however, com-

plex post-processing algorithms are generally needed. Various

examination protocols are possible in this context. On the one

hand, it is possible to acquire a set of images from a single angle

position to identify the different breathing/heart phases in these

images and repeat this process from several angles. The secondpossibility consists of continuous image acquisition with a high

frame rate at a constant rotation speed and the rearrangement of 

single projections of the resulting dataset according to the heart/

breathing phase. In this setup, generally several scans are

performed in a row [94], then the images acquired during breath-

ing excursions are discarded and the remaining images are

grouped by heart phase to new raw datasets. Fig. 10 shows a data-

set acquired with a volume-CT scanner using this last method to

determine left ventricular function, myocardial thickness, and lung

volume in mice, to name a few.

 3.3.2. Thoracic imaging 

The successful implementation of thoracic   lCT allows the

detection of pulmonary lesions in a murine adenocarcinoma model[2]. In this study, Li et al. were able to detect lung tumors P1 mm

Fig. 8.   Images (A + B) show volume rendering datasets of the circle of Willis of a

BALB/c mouse, (C + D) of a C57BL/6 mouse with visible anterior cerebral a. (ACA),

middle cerebral a. (MCA), internal carotid a. (ICA), posterior cerebral a. (PCA),

superior cerebellar a. (SCA), posterior communicating a. (PcomA) and basilar artery

(BA). In BALB/c mice the posterior circulation is mainly supplied over the PcomA,

whereas in C57BL/6 mice the related territory is supplied by the BA and SCA, which

can lead to different infarction territories in murine stroke models corresponding tothe difference shown in vessel anatomy.

Fig. 9.   Volume rendering of ungated murine datasets using the blood-pool contrast

agent Fenestra VC

. (A) shows abdominal vasculature and (B) shows thoracicvasculature. Veins and arteries are equally visible in both datasets.

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in diameter in unenhanced scans. Even though a sharp cutoff for

the tumor border could not be defined, it was still possible to dis-

tinguish the lesion from abutting vascular structures with the help

of other slice orientations. In a more recent study, it was possible todelineate pulmonary tumors of 0.85 mm in vivo in a nude mouse

model [95]. One of the latest in vivo studies of mice attained de-

tailed perceptibility of lung tumors of 500 lm using respiratory

gating [96].

Other studies describe the capability of  lCT for the monitoring

of lung fibrosis in mice after intratracheal administration of 

bleomycin   [97]. The fibrotic lung segments present themselves

with a rising compaction of lung parenchyma. In another model,

emphysema was produced in mice via tracheal instillation of pan-

creas elastase and the typical changes in lung architecture could be

detected   [98]. The differences in regional lung ventilation were

non-invasively imaged in Wistar rats via xenon gas inhalation

lCT [99].

 3.3.3. Cardiac imaging 

Hypercholesterolemia and arteriosclerosis can produce oxida-

tive stress, a dysfunction of coronary arteries and myocardial

ischemia, which accompany the expression of growth factors and

can lead to myocardial neovascularization. Using high-resolution

lCT ex vivo, Zhu et al. demonstrated that it was possible to image

the related changes in myocardial micro-vasculature in swine

[100]. In this quantitative analysis, the subendocardial spatial den-

sity of microvessels with a diameter under 200 lm, at an imaging

resolution of 40 lm was significantly higher in swine suffering

from hypercholesterolemia compared to controls.

For in vivo imaging of the heart in rat and mice, projections

must be assigned not only to different breathing phases, but also

to higher frequency heart actions. Cardiac gating in small animalshas been successfully described in the literature in terms of the

application of prospective and retrospective gating techniques

[84,101]. Using pulsed X-ray tubes as done by Badea et al.  [84], a

10 ms time window per frame is possible. Temporal resolution of 

a dedicated X-ray detector should be high enough to display oneheart cycle in a set of images. Most detectors in use do not exceed

a frame rate of 30 fps. The implication here is that at frame rates of 

30 fps the end-diastolic and end-systolic phase can be identified in

animals with a heart rate of up to 300 bpm. Images that we pro-

duced under such conditions via retrospective intrinsic gating in

mice are presented in Fig. 10.

To summarize, in vivo cardio-CT is well established as a valu-

able method of non-invasive investigation in heart pathology mod-

els as well as for the phenotyping of genetically modified small

animals.

 3.4. Imaging of abdominal organs

lCT imaging of abdominal organs in small animals is increas-ingly used as the availability of scanners has greatly improved. In

this context, the examination of parenchymatous organs such as

liver, spleen and kidney is of high interest, whereas most studies

focus on oncological problems and the investigation of the kidney

function.

 3.4.1. lCT of parenchymatous upper abdominal organs

Relatively long scanning times, in the range of minutes, motion

blurring caused by breathing movements, and the small blood

volume of animals make the evaluation of subphrenic abdominal

organs, like the liver, difficult when using conventional iodinated

contrast agents. As a result, for the imaging of parenchymatous

upper abdominal organs, liver-specific contrast agents are used

on a regular basis. For example, the iodinated contrast agentDHOG (1,3-bis-[7-(3-amino-2,4,6-triiodophenyl)-heptanoyl]-2-oleoyl

Fig. 10.   Datasets of the murine heart after retrospective intrinsic gating. Images (A + B) show a short axis view in MIP mode with (A) representing end-systolic and

(B) end-diastolic phase. In (D) a long axis view of the heart in end-diastolic phase with clearly distinguishable left ventricle (LV), right ventricle, atriums and aortic arch (AA).

(C) shows a volume rendering with pulmonal tissue visible in light blue and trachea as well as bronchioli in red. (Intrinsic gating in cooperation with Q. Xie, Experimental

Radiation Oncology, Medical Faculty Mannheim, and S. Bartling, German Cancer Research Centre (DKFZ).)

S.J. Schambach et al./ Methods 50 (2010) 2–13   9

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glycerol; Fenestra LC; Art Technologies, San Diego, CA), which is

internalized by hepatocytes via the ApoE-receptor and leads to a

relevant enhancement of liver contrast after 1–2 h   [86,88,102].

DHOG allows for the delineation between healthy, enhancing

hepatocytes and non-enhancing neoplasmatic cells [103] and thusthe non-invasive quantification of liver metastases. The burden-

some ‘‘second look” procedure used in older experimental setups

can, therefore, be abandoned. Furthermore, liposomes are ingested

by phagocytic cells in the spleen, and the red pulp which is

populated mostly by erythrocytes and macrophages. This ingestion

creates a hyper-dense imaging effect [44]. As the half-life of DHOG

can range from several days up to 2 weeks, it may offer the possi-

bility to image the tumor growth repeatedly without the need for

repeated injections, or at least to reduce the dose of the adminis-

tered contrast agent in repeated scans.

The smallest neoplasms that could be confirmed in a respira-

tory-gated DHOG enhanced  lCT scan have been reported to be in

the range of 250–300 lm [44]. However, Fig. 11  shows a murine

liver interspersed with metastases with diameters of less than100 lm scanned about 2.5 h after i.v. administration of 400ll

Fenstra LC.

 3.4.2.  lCT of the kidney

Up to 10 years ago it was difficult to geometrically relate the 3D

anatomical complexity of renal vasculature with the related tubu-

lar sections of the nephron because of methodological limitations.

A recent review on the use of lCT for the evaluation of renal micro-

structural changes addresses the paucity of imaging publications

citing only 16 articles  [104]. Most of the cited articles evaluated

anatomy ex vivo to compare 3D anatomy to histological slices from

human specimens.

Likewise, only a few publications describe the use of   lCT to

evaluate renal structures in animals. Using  lCT, Fortepiani et al.showed that the blockage of nitrogen-oxide synthesis (an experi-

mental approach to elevate blood pressure) leads to a reduction

in renal blood flow and that administration of an AT1-receptor

antagonist inhibited the effects of this blockage. To evaluate vessel

changes, the kidneys were perfusion fixed in situ and perfused

with radiopaque silicon. Furthermore, an elevated heterogeneity

of glomerular volumes in the renal cortex of rats suffering from

diabetic nephropathy was confirmed via  lCT [105].

Even fewer studies on the use of   lCT for in vivo imaging in

small animals exist. A study by Almajdub et al. describes an

in vivo imaging procedure for mouse kidney anatomy evaluation

using contrast-enhanced high-resolution  lCT [106]. They demon-

strate that contrast-enhanced  lCT enables accurate in vivo mea-

surement of kidney volume, length and thickness in mice andreport reference parameters for four strains.

Other authors applied  lCT of the kidneys in small animals to

evaluate liposomal blood-pool contrast agents by ruling out renal

excretion [43,89].

As an example of renal in vivo  lCT imaging, Fig. 12 presents ascan of the renal pelvis and the ureter of a mouse after contrast

agent administration is presented.

 3.4.3. Gastrointestinal tract 

Colon carcinoma is one of the most frequent tumors in Western

society. Various animal models exist to mimic this disease andlCT

is applied successfully in this research field as well. For example, in

a study by Durkee et al., virtual colonoscopy was performed anal-

ogous to the examination of colon polyps in humans. After nega-

tive contrasting by air insufflation in APC-min-mice, colon polyps

could be visualized and measured [107]. In a subsequent histolog-

ical examination, a high correlation between identified tumor vol-

umes via   lCT and histology was found. Using various positivecontrast agents, it was also possible to image spontaneously

Fig. 11.  MIP of the murine liver contrasted with Fenestra LC in transversal (A) and coronal (B) view (1200 projections, scan time 40 s, Field of View 3.5 3.5 cm). White

arrows point to hypo dense round structures with a diameter of  100 lm, in the sense of metastases.

Fig. 12.  Volume rendering of lCT scan of a C57BL/6 mouse in vivo after injection of 

the blood-pool contrast agent Fenestra VC, acquired without cardiac or pulmonary

gating. The numbers refer to the abdominal vena cava (1), the renal vein (2), right

iliac vein (3), spleen (4), kidney (5), and the ureter (6).

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emerging colon polyps in Cdx2+/ mice, as well as in mice bearing

azoxymethane-induced polyps  [108].   Fig. 13   shows a 3D virtual

colonoscopy dataset of a mouse, as well as a two-dimensional

X-ray with a double-contrasted colon performed on a volume-CT.

 3.4.4. Quantification of body and organ fat content 

The estimation of body and organ fat content is important be-cause of a high quantity of metabolic syndromes with a related

alteration in the percentage of body fat. Although various other

methods allow for the quantification of body fat,   lCT offers a

non-invasive and elegant possibility to quantify not only body fat

volume, but the degree of fatty degeneration of distinct organs as

well [109].

 3.5.  lCT of cerebral structures

Several ex vivo studies analyzing cerebral microcirculation and

anatomical details of small rodents have been performed, as they

allow longer scans on samples to achieve a higher SNR and higher

spatial resolution after appropriate treatment of the tissue [110–

113]. For example, it was possible to distinguish the white andgraymatter, as well as tumor tissue in mouse brain samples after they

were allowed to absorb iodinated contrast agent [114].

While cranial CT is a routine method in clinical CNS examina-

tions, only a few publications describe the use of   lCT for CNS

examination in small animals. In a study by Newcomb et al., the

size of intracerebrally growing invasive gliomas in mice was mea-

sured using a clinical CT scanner [115]. The image quality was suf-

ficient to see the contrast-enhancing tumor parts with moderate

resolution. Engelhorn et al. were the first to report the use of a

lCT for double dose contrast-enhanced in vivo imaging of xeno-

grafted rat brain glioma   [116]. They found a good correlation of 

lCT- and 3.0T clinical MRI-derived tumor volumes compared to

histology. Another in vivo study was conducted by Balvay et al.

who examined the microcirculation of gliomas in Wistar rats viadynamic contrast-enhancedlCT, although they used a synchrotron

radiation source [117]. To our knowledge, to date there are no pub-

lications exploring intracranial tumors in mice using   lCT in an

in vivo setting.

4. Concluding remarks

4.1. Summary and future directions

With the increased availability and user-friendliness of   lCT,

there has been an increased opportunity for preclinical research.

Simple operability, fast scanning protocols, a significantly higher

temporal and spatial resolution, plus lower acquisition costs and

maintenance, all prove beneficial compared to high-field small ani-mal MRI. However, with a view to MRI,  lCT can also be seen as a

complementary, additional and adjuvant technique rather than a

competitive one. Disadvantages in comparison with MRI, are the

lower soft tissue contrast, and the particularly high radiation dose.

For example, measurements with thermoluminescent dosimeters

implanted in mice yield a radiation dose of 10–50 cGy per CT scan

[10], whereas with very small source–object distance dose levels of 

up to 5 Gy with dermal depilation after 2–3 weeks are reported[16].  Therefore, the cumulative dose can be very high, especially

in longitudinal studies, as such the radiation associated biological

effects depend not only on radiation dose but also on the applica-

tion time frame. As a result, the radiation dose needs to be consid-

ered in the planning of oncological in vivo studies using  lCT. A

cumulative whole body radiation dose of 50 cGy within 7 days

leads to a significant increase in the incidence of ovarian cancer

in BALB/c mice [118], and a dose of 1 cGy to a significant reduction

of tumor volume in lymphoma mouse models [119]. A whole body

dose of 50 cGy decelerates the progression of diabetes type I in

non-obese diabetic mice [120]. Based on these findings, the chal-

lenge in  lCT imaging will be the acquisition of valuable datasets

at much lower radiation doses. These drawbacks can be minimized

using novel contrast agents, and up-to-date low-dose l

CTs  [121]

with newer, more sensitive detectors and intelligent scanning

protocols.

Along with an increase in the employment of  lCT, the growing

availability of PET-CTs can be expected from various companies

such as Bioscan, GE, GMI or Siemens (for details, see the homepag-

es listed in  Table 1). PET offers extremely high sensitivity in the

detection of targeted neoplasms at a low resolution, which is coun-

terbalanced by an image overlay of a high-resolutionlCT scan that

is acquired in the same session. The fusion of lCT and fluorescence

imaging could be of interest as well and would obviate the use of 

radioactive tracers. However, this fusion could only be applied to

small animals such as mice or for superficial processes because

of the low light intensity and tissue penetration of fluorescent

light. The combination of two procedures like PET or fluorescence

imaging and CT does not necessarily mean the acquisition of a

new combined scanner but can be achieved via a subsequent fu-

sion of datasets recorded with separate scanners.

Starting from an ongoing optimization of scanner equipment

such as more sensitive detectors with even higher temporal and

spatial resolution, reduced radiation doses, and further improve-

ments in software protocols, a long-term increase in the use of 

lCT in preclinical research can be expected. Additionally, improve-

ments in gating algorithms and iterative low noise reconstruction

methods may lead to an increasing acceptance and availability of 

lCT methods.

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