ORIGINAL PAPER

Possibilities and limits of X-ray microtomography forin vivo and ex vivo detection of vascular calcifications

A. A. Postnov Æ P. C. D’Haese Æ E. Neven ÆN. M. De Clerck Æ V. P. Persy

Received: 19 September 2008 / Accepted: 25 March 2009 / Published online: 11 April 2009

� Springer Science+Business Media, B.V. 2009

Abstract In the present paper, vascular calcifica-

tions due to chronic renal failure in rats are studied by

X-ray microtomography (l-CT). Although l-CT is

traditionally used as an imaging technique, a quan-

titative analysis of data obtained by in vivo and ex

vivo l-CT is described and discussed. By comparison

with traditional destructive methods, such as histo-

morphometry and atomic absorption, the detection

limits for calcium were determined in living rats and

in extracted aortas. l-CT proved to be an effective

non-invasive imaging technique allowing non-

destructive quantification of ectopic calcifications.

Keywords Vascular calcifications �Chronic renal failure � X-ray microtomography �Image analysis

Introduction

X-ray microtomography (l-CT), a downscaled ver-

sion of computed tomography (CT) is being

increasingly used in animal research since the late

1990s [1–3]. In laboratory rodents this technique

allows non-invasive in vivo and ex vivo imaging of

body structures, ranging from ultra soft tissues with

densities close to air such as lungs [4] up to dense

metal implants [5]. Recently, l-CT scanning has been

used to examine lung cancer [6], lung emphysema

[7], osteoporosis [8], cochlear implants [9] as well as

in dental research [10]. Visualization by laboratory

tabletop l-CT systems and subsequent image analysis

are most successful in calcified tissues like bones due

to the X-ray energy range, which is used. Moreover,

it was shown previously that this method could also

be applied to detect and to quantify ectopic calcifi-

cations in the vasculature of rats with chronic renal

failure (CFR) [11]. l-CT is of particular interest for

long-term experimental follow-up and intervention

studies, as it allows the assessment of changes that

take place over time without the need of sacrificing

the animals [11, 12].

The main purpose of the present study is to

demonstrate and to evaluate the analytical perfor-

mance of l-CT in the in vivo and ex vivo detection

and quantification of vascular calcifications in aortas

of an adenine-induced chronic renal failure rat model.

In addition, emphasis is put on the correlation

between quantitative data obtained by l-CT and the

tissue calcium content assessed by atomic absorption

spectrometry (AAS) as well as histomorphometric

scoring of Von Kossa-stained sections, which are still

considered as the ‘‘gold standard’’. The possibilities

A. A. Postnov (&) � N. M. De Clerck

Laboratory of Microtomography, University of Antwerp,

Universiteitsplein 1, Campus Drie Eiken, Gebouw N,

lokaal 010, 2610 Antwerp, Belgium

e-mail: andrei.postnov@ua.ac.be

P. C. D’Haese � E. Neven � V. P. Persy

Laboratory of Pathophysiology, University of Antwerp,

Antwerp, Belgium

123

Int J Cardiovasc Imaging (2009) 25:615–624

DOI 10.1007/s10554-009-9459-9

and limits of l-CT in the detection and quantitative

analysis of vascular calcifications are discussed in

detail.

Methods

Animal procedures

In this study chronic renal failure was induced in 50

male Wistar rats of 250 g (Iffa Credo, Brussels,

Belgium) by feeding them an adenine diet. As we

aimed to use in vivo l-CT for selecting positive

animals out of non-calcified rats, all animals were

scanned for the first time after 6 weeks of CRF. Rats

showing vascular calcification were further followed

until week 9, at which time they were scanned for the

second time. Negative animals were sacrificed. This

study set-up is summarized in Fig. 1. Eight animals

that died spontaneously during the experiment also

underwent histomorphometrical studies. Results from

47 animals from which l-CT and histological data

are available are reported here (three rats died before

scanning was started). All experimental procedures

were carried out in accordance with the NIH guide

for the care and use of laboratory animals No. 85-23

(1985) with approval of the University of Antwerp

Ethics Committee.

The animals were housed in pairs, in plastic cages,

with a 12 h light/dark cycle and with tap water and

food available ad libitum. CRF was induced by a diet

containing 0.75% adenine (0.92% P and 1.0% Ca) for

4 weeks. This has been shown to induce a stable,

moderate to severe renal function impairment in rats

[13]. During further follow-up, rats received a

pelleted diet containing 1.06% Ca and 1.03%

P. Diets were custom made by SSNIFF Spezialdiaten

(Soest, Germany).

The adenine model of CRF is characterized by a

stable, moderate to severe renal function impairment,

with serum creatinine values increasing from

0.45 ± 0.06 mg/dl in normal animals to 2.61 ±

0.52 mg/dl after 4 weeks of CRF and remaining

stable afterwards. Although the degree of renal

failure and its associated biochemical characteristics,

such as increased PTH and low 1.25(OH)2 vitamin D,

are similar in all animals, vascular calcification is

only observed in about half of the animals [14].

Similar results were obtained by Price et al. [15].

Mineral deposits in the aorta and large arteries were

present by week 6 after induction of CRF and did not

increase significantly afterwards [14].

Microtomography

An in vivo X-ray l-CT scanner (Skyscan-1076,

Kontich, Belgium) was used. This instrument is

equipped with a 100 kV/100 lA micro focus tungsten

X-ray tube with a 5 lm focal spot size. A 10-mega-pixel

CCD camera (Hamamatsu ORCA-HRF) was used as

an X-ray detector. The smallest pixel size on the

radiography projection is 9 lm. Binning of 4 or 16

camera pixels in one image pixel results in 18

and 35 lm image pixel sizes providing better signal-

to-noise ratio.

For our experiments a 35-lm image pixel was

selected. A 25-lm titanium filter and a 90 kV X-ray

tube voltage were chosen for the in vivo scanning

whilst an aluminum filter and 100 kV were applied

for the ex vivo experiments. Application of a Ti filter

removes the softest part of X-ray spectra that would

be completely absorbed in the animal and hence

reduces radiation dose received by the rat. The Al

filter leaves the restricted high-energy part of

Fig. 1 Flow diagram of the experimental set-up

616 Int J Cardiovasc Imaging (2009) 25:615–624

123

Bremsstrahlung generated by the tungsten X-ray tube

and therefore reduces beam hardening which is

important for quantification.

In vivo detection of calcifications

For in vivo studies, rats anesthetized with 35 mg/kg IV

sodium pentobarbital (Nembutal, Ceva Sante Animale,

France) were placed on their back in the 60 mm wide

animal bed of the scanner. These conditions allowed

scanning of 18 mm axial length of the thorax during a

20 min exposure time. The scanned region started at

the top of the fourth rib, with radiographic X-ray

images of the scanned region saved as an anatomical

reference for follow-up scans. These conditions proved

to be safe for the animals and allowed serial scanning

with some time interval. The temporal evolution of the

shape and volume of the mineral precipitate was

analyzed from tomographic slices [11].

Transverse virtual cross-sections were recon-

structed from the raw dataset by the Feldkamp cone

beam algorithm [16]. In each rat, a region containing

the aorta was reconstructed. The presence of calci-

fications was evaluated de visu and scored as follows:

-: negative, ?: focal calcification or partially

circumferential dense delineation of the aortic wall

in at least two cross-sections, ??: circumferential

calcification of the aortic wall, ???: excessive

circumferential calcification with regional increases

in wall thickness.

Tissue calcium content (AAS)

The proximal part of the abdominal aorta was

weighed with a precision balance and digested

overnight in 65% nitric acid at 65�C. Subsequently,

the tissue digests were adjusted to a final volume of

2.5 ml with milliQ water. Tissue calcium content was

measured by flame atomic absorption spectrometry

(Model 3110, Perkin-Elmer) after appropriate further

dilution of the samples in 0.1% lanthanum nitrate to

avoid chemical interferences. Results are expressed

as mg Ca/g wet tissue weight.

Histomorphometric measurement of Von Kossa

positivity

Prior to embedding in paraffin, formalin-fixed aortic

tissue was sectioned perpendicular to its length axis

into 1–2 mm long segments. Segments from the

aortic arch (6–10 per animal) and thoracic aorta

segments (10–19 per animal) were embedded upright

in two separate paraffin blocks. Sections each 4 lm

thick were stained with Von Kossa’s method to

visualize calcium-containing precipitates and coun-

terstained with hematoxilin and eosin.

Vascular calcifications were evaluated histomor-

phometrically on digital images obtained with an

Axiocam HRm (Carl Zeiss Imaging Solutions)

coupled to a Leitz-Aristoplan microscope (WildLeitz

Co., Rockleigh, NJ, USA) with the Axiovision 4.5

image analysis software (Carl Zeiss Imaging solutions,

Germany). Sections were analyzed at a magnifica-

tion of 62.59 with two different color separation

thresholds, one to measure the area fraction in

percent of the slide covered with tissue, and another

to measure the area fraction in percent of Von Kossa

positivity. The possibility for manual user interven-

tion was built into the program to eliminate artifacts

(contaminations, other tissues that should not be

taken into account) outside the aorta, which falsely

increase the tissue area, and tissue folds, which can

give rise to overestimation of the calcified tissue

area. The absolute areas of tissue and calcified tissue

were summed for each animal and the ratio was

expressed as area fraction in percent of aortic

calcification.

l-CT measurements of extracted tissue

Extracted aortas were fixed in neutral buffered

formalin for 90 min at room temperature, subse-

quently rinsed in 70% isopropanol and stored at 4�C

until l-CT scanning was performed. The same

material was then processed for paraffin embedding

and microscopic analysis.

In contrast to the in vivo situation, where motion

artifacts are unavoidable and where scanning times

should be kept relatively short, Ca density and

mineral content can be estimated in isolated fixed

calcified aortas.

A map of linear attenuation coefficients recon-

structed from radiographic projections is used to

define these parameters. The method is fully

described in [17].

Atomic absorption spectrometry, being a com-

pletely independent analytical method was applied to

validate observations by l-CT.

Int J Cardiovasc Imaging (2009) 25:615–624 617

123

The density measured by l-CT is not equivalent to

the Ca concentration measured by AAS because it

only relates to the calcified part of the aorta, whereas

AAS takes into account the total amount of tissue;

i.e., calcified as well as non-calcified regions of the

aorta. For example, one small but dense deposit in the

aorta will have a high density in l-CT measurement

whereas with AAS the Ca concentration of this

deposit will be averaged over the whole non-calcified

aortic mass and will only marginally contribute to the

integral density value assessed by l-CT.

Results

In Table 1 the results of different methods used to

evaluate vascular calcification are summarized: i.e.,

traditional post-mortem techniques (histomorphome-

try and AAS), in vivo l-CT and ex vivo l-CT.

Animals are sorted by decreasing Ca content mea-

sured by AAS. In the in vivo experiment a total of 47

animals were scanned to collect sufficient statistical

data to establish the analytical performance of l-CT

in terms of detection limits, precision and accuracy.

Out of the total number of rats, observations from 32

animals were reported in Table 1. Rats that died

before the end of the experiment and did not undergo

all measurements were excluded, with exception of

five animals (#1, 2, 5, 6 and 10) that showed a high

calcification rate during in vivo l-CT studies. Ani-

mals where no calcification was detected by any of

the methods used, were also omitted from the table.

In vivo detection of early calcifications

Figure 2 shows a sagittal virtual cross-section recon-

structed from a set of radiographic projections. The

calcified wall of the aorta is much denser than the

surrounding soft tissues. If no calcifications are

present, the aorta cannot be distinguished separately.

In this and all following cross-sections dark colors

correspond to high attenuation, and light colors

correspond to low attenuation.

In one rat, an aneurysm was observed as illustrated

in Fig. 3, where a transverse virtual cross-section is

shown.

The results in Fig. 4 illustrate that aortic calcifi-

cations can appear in different shapes and degrees

such as a partially calcified aorta, a completely

calcified circle or excessive calcification. These latter

observations were confirmed by histology as shown

in Fig. 4.

To determine the detection limits of in vivo l-CT,

scanning was started at week 6 of treatment when

stable chronic renal failure is present and vascular

calcification is at its height. Although all rats suffer

from chronic renal insufficiency after being treated

with the 0.75% adenine diet for 4 weeks, calcifica-

tions were not observed in all of them. Indeed, in this

animal model the percentage of animals with calci-

fications varies around 50–60% [11]. Fourteen

animals were scored positive by l-CT scanning.

Additional assessment of vascular calcifications by ex

vivo l-CT, AAS and histomorphometry (Von Kossa

staining) indicated that the total number of affected

animals was 20. Thus, using in vivo l-CT scanning,

70% of the animals having calcifications could be

detected before sacrifice.

The detection limit of in vivo l-CT analysis was

determined by comparison to ex vivo techniques

(AAS, histomorphometry and ex vivo scanning). The

minimum amount of calcium that could be detected

in a scanned region in a live rat corresponded to

50 lg (see animals #13–15). The minimum volume

of calcified tissue detected in a live animal was

1.2 mm3 (animal #13). Measurement of the bulk Ca

concentration in the samples scored positively in vivo

is generally higher than 5 mg Ca/g wet weight tissue.

The lowest degree of calcification detected with l-CT

corresponded to a bulk Ca concentration of 4.55 mg/

g (animal #15). This value is approximately 20 times

higher than that noted in non-calcifying animals with

abnormal renal function. Hence slightly affected

animals can be missed with in vivo l-CT. On the

other hand, our follow-up study in rats indicates that

calcification, once started, develops rapidly and that

Ca concentrations in general quickly reach values up

to 30 mg/g. In one animal calcifications could not be

detected by l-CT despite the relatively high Ca

content measured by AAS (11.49 mg/g, animal #9).

Ex vivo quantitative analysis of calcification

In contrast to in vivo l-CT, ex vivo scanning proved

to be a much more sensitive method. In preserved

material, calcification becomes visible if the volume

of a calcified unit exceeds 0.025 mm3 (animal #19).

However, the density of calcifications (minimum

618 Int J Cardiovasc Imaging (2009) 25:615–624

123

Table 1 Comparison of in vivo and ex vivo l-CT with AAS and histomophometric analysis of Von Kossa-stained microscopy

sections

Post-mortem techniques X-ray microtomography

Animal

#

AAS wet

tissue

(mg/g)

Von

Kossa

(area %)

Histology

scoring

Visual

l-CT

(in vivo)

Visual

l-CT

(ex vivo)

Volume

of calcified

tissue, l-CT, mm3

Ca, l-CT (lg) Ca density l-CT (mg/cm3)

1 31.38 21.33 ? ?? NA NA NA NA

2 28.96 26.39 ? ??? NA NA NA NA

3 26.28 10.4 ? ? ? 5.989 330 55.3

4 22.38 22.65 ? ??? ? 30.321 1,990 65.8

5 19.98 20.35 ? ?? NA NA NA NA

6 19.26 22.95 ? ?? NA NA NA NA

7 16.94 17.83 ? ?? ? 27.479 1,520 55.3

8 13.02 9.334 ? ?? ? 16.121 650 40.6

9 11.49 3.896 ? - ? 0.784 23 29.05

10 10.66 17.98 ? ?? NA NA NA NA

11 8.55 13.25 ? ? ? 8.999 350 39.2

12 8.15 10.24 ? ? ? 11.442 400 35.35

13 6.15 10.01 ? ? ? 3.177 65 20.65

14 5.46 1.313 ? ? ? 1.276 50 39.9

15 4.55 5.14 ? ? ? 1.752 61 34.65

16 4.16 1.107 ? - - 0.019 0.35 18.55

17 3.68 1.767 ? - ? 0.137 2.6 19.25

18 3.11 4.552 ? - ? 0.174 3.6 21

19 2.62 1.404 ? - ? 0.025 0.45 18.55

20 2.04 0.512 ? - ? 0.093 2.6 28.35

21 1.57 0.012 - - - 0.018 0.21 12.6

22 1.4 2.479 ? - ? 0.060 1 17.15

23 0.98 0.003 - - - 0.005 0.035 6.65

24 0.8 0.08 - - - 0.003 0.01 3.15

25 0.76 0.011 - - - 0.010 0.14 14

26 0.37 0.002 - - - 0.006 0.07 8.4

27 0.33 0.012 - - - 0.001 0.06 6.3

28 0.31 0.002 - - - 0.014 0.175 12.25

29 0.26 0 - - - 0.006 0.035 5.95

30 0.23 0 - - - 0.001 0.00035 0.35

31 0.19 0.002 - - - 0.020 0.315 15.05

32 0.19 0.009 - - - 0.025 0.315 13.3

Animals are sorted by the decrement of Ca content measured by AAS

NA not analyzed

Scoring system for in vivo visual l-CT is the following: -, negative; ?, focal calcification or partially circumferential dense

delineation of the aortic wall in at least two cross-sections; ??, circumferential calcification of the aortic wall; ???, excessive

circumferential calcification with regional increases in wall thickness. Volume, amount of calcium and calcium density were

calculated from ex vivo l-CT data

Int J Cardiovasc Imaging (2009) 25:615–624 619

123

15 mg Ca/cm3) also determines what can be detected

by ex vivo l-CT.

Very small (less than 0.025 mm3) but dense

calcifications that were detected by ex vivo l-CT

could not be observed by any other method used in

this study. This was the case in animals (#28, 31 and

32).

AAS and histomorphometry based on Von Kossa

staining

Atomic absorption spectrometry analysis of the

proximal part of the abdominal aorta resulted in

typical values from 31 mg Ca/g of wet tissue for

heavily calcified animals down to 0.16–0.19 mg Ca/g

for animals with undetectable calcification. Morpho-

metric analysis of Von Kossa staining measures the

area taken by calcified tissue compared to the total

area of aorta tissue. Here, 20% positivity corresponds

to the heaviest calcifications. Representative exam-

ples are shown in Fig. 5. Results from histological

scoring by light microscopy are presented in Table 1

(third column). The smallest detectable Von Kossa

positive area for calcified aorta is 0.5%. Aortas with a

score \0.5% can be considered as not calcifying.

Figure 6 illustrates the difference in detection

limits between histology, AAS and l-CT.

When approaching detection limits, histology

remains more sensitive when calcification is present

as tiny particles that are much smaller than l-CT

resolution (Fig. 6a, b). Animal #16 is the only excep-

tion that scored positively by histomorphometry but

negatively by l-CT analysis, which most probably is

due the fact that the calcified volume was too small.

Fig. 2 Representative sagittal virtual cross-section of a fully

calcified aorta reconstructed from in vivo l-CT. Notice (arrow)

the calcified walls of aorta. Non-calcified parts cannot be

distinguished from the surrounding tissue

Fig. 3 Transversal virtual cross-section of an in vivo scan

showing a calcified aorta with aneurysm

Fig. 4 Virtual cross-section illustrating different types of

calcifications. Non-calcified parts remain invisible, the arrowsindicate partially calcified aorta and completely calcified circle;

the insert shows excessive calcification

620 Int J Cardiovasc Imaging (2009) 25:615–624

123

This situation is highlighted by Fig. 6c, d. On the

other hand, l-CT analyzes and visualizes the entire

sample and can detect individual spots that are too

small to give contribution to AAS signal and can

easily be missed on histology (Fig. 6e). Visualization

by l-CT also helps to detect contaminations (Fig. 6f)

that are present in some preparations and can confuse

AAS measurements.

Discussion

In line with previous studies [11], the present report

clearly shows that vascular calcification in rats with

chronic renal failure (CRF) can be detected and

visualized by in vivo X-ray microtomography. By

validation and comparison with standard ex vivo

techniques, the detection limits for both in vivo and

ex vivo l-CT were determined.

The results presented in Table 1 summarize

observations by traditional techniques and by l-

CT. Data indicate that although the typical total Ca

concentration of non-calcified tissue is around

0.18 mg/g (AAS), only those samples having a total

Ca concentration above 1 mg/g were confirmed

positive by histomorphometric analysis. Whereas

AAS and histomorphometry were applied to differ-

ent tissue samples, only one animal (#21) had

significantly different calcification and could have

been missed if only histological scoring had been

performed.

Von Kossa staining is a very sensitive dying

technique, but histomorphometric measurement of

the sections is influenced by both staining and cutting

artifacts. Hence, the animals that were considered

positive actually have a positive area that exceeds

0.5%.

Out of 20 animals identified as having calcifica-

tions by ex vivo analysis, 14 were also found positive

by in vivo l-CT corresponding with a successful

detection of calcifications in 70% of the cases. The

only animal showing a high Ca content as assessed by

AAS, which was not detected in vivo, was animal #9

(see Table 1). This can be explained by a focal

distribution of the calcifications. The analysis of

extracted aortas proved that the estimated calcified

volume in animal #9 is 0.78 mm3 being below the in

vivo detection limit (1.2 mm3 as detected in animal

#14). This volume is relatively large because of aorta

contractions. The lower the calcification level is, the

more is the amplitude of the contractions; so calcified

area should be high enough to overcome the blurring

Fig. 5 Histological images

of different degrees of

calcification (von Kossa

staining). a Aorta cross-

section without calcification

(0%), b calcified spot

(1–3%), c completely

circumferential calcification

(*10%, compare to Fig. 3),

d excessive calcification

(*20%)

Int J Cardiovasc Imaging (2009) 25:615–624 621

123

effect. Data in Table 1 further demonstrate that for a

reliable detection of vascular calcifications, Ca con-

centration/density in the sample should be high

enough together with the presence of a sufficiently

large volume of calcified tissue. Strict border condi-

tions are difficult to define because of the limited

number of experimental data. But it should be taken

into account that the number of photons that form the

shadow projections and hence lower signal-to-noise

ratio restricts accuracy of in vivo analysis. In our

study the lowest Ca density detected in vivo was

20.65 mg/cm3 for animal #13 (volume of calcified

tissue was 3.2 mm3).

The possibility of early in vivo detection of

calcification is of particular interest for follow-up

and intervention studies in which for example the

effect of therapeutics on the reversibility of vascular

calcifications has to be evaluated. In such experi-

ments, in vivo l-CT analysis on the one hand allows

withdrawal of non-calcifying animals, and follow-up

of calcifying animals on the other hand, as previously

demonstrated in a feasibility study [11].

Fig. 6 Demonstration of the difference in detection limits

between histology, AAS and l-CT: a ‘‘sand-type’’ Von Kossa

positivity. Sizes of each grain are too small to be detected by

l-CT. Nevertheless, the total amount of calcified tissue is

enough to be visualized by histology and add to AAS signal.

b Magnified image of the affected area. Notice a lot of tiny

calcifications. c Small calcified spot that is detected histomor-

phometrically, but can be considered as statistical noise during

l-CT analysis. It also contributes marginally to AAS.

d Magnified image of individual spot. e l-CT detects a single,

focal Ca deposit that brings a very small contribution to AAS

signal and can be easily missed in histology due to sampling

bias. f l-CT image showing a dense focus outside the aorta

wall. This calcification is excluded during histomorphometry

as well as during quantification of l-CT cross-sections, but can

add to AAS signal

622 Int J Cardiovasc Imaging (2009) 25:615–624

123

The fact that two of three calcified animals were

detected in vivo means that the major part of affected

animals could be selected for follow-up studies and

we believe it is a positive contribution in biomedical

research. These animals can serve as their own

control and dramatically reduce total number of rats

involved in the experiment.

When comparing in vivo l-CT to AAS, different

distribution patterns of calcifications (heteroge-

neously localized dense spots vs. an homogeneous

distribution) leads to different detection limits of in

vivo l-CT. A given density of e.g., 5 mg Ca/g tissue

measured by AAS can easily be detected with in vivo

l-CT in case calcifications are present as high density

focal spots whereas measurement will not be possible

when the same amount of Ca would be uniformly

distributed over the whole tissue section. It might be

the reason why an animal #15 with an aortic Ca

concentration as low as 4.5 mg/g calcifications could

be detected in vivo.

Selection of the parameters for early in vivo

detection by l-CT is complicated by the following

factors: radiation dose, scanning time and motion

artifacts. Radiation dose restricts scanning time and

consequently signal-to-noise ratio that leads to a

limited detection level, which obviously remains

lower than in ex vivo situations. Motion artifacts are

inherent to a living animal and are always a limiting

factor for in vivo l-CT. Three main motion types can

be distinguished in an anesthetized animal: cardiac

contractions, respiration and motility. In view of this,

one will always be confronted with some blurring and

artifacts in reconstructed cross-sections.

Finally, the detection limit of in vivo l-CT in the

detection of vascular calcification was found to be

equivalent to a Ca concentration of 5 mg/g measured

by AAS. Further improvement of the limit of

detection can be achieved by either increasing the

acquired signal, for example, by increasing scanning

time, and/or reducing blurring resulting from motion

artifacts.

In ex vivo studies the sensitivity of the method

improves dramatically. Results of visual inspection of

reconstructed cross-sections of ex vivo studies corre-

lated with histological observations with the exception

of one animal #16. The smallest detected volume of

calcified tissue detected by ex vivo l-CT was

0.025 mm3 and the smallest amount of Ca was

0.45 lg (animal #19). Since there is only one animal

that showed discrepancies between traditional meth-

ods and l-CT, we can estimate the detection accuracy

ex vivo to be [95%. Signal-to-noise ratio can be

improved by increasing scanning time while the flat

field correction of X-ray CCD detector remains valid.

Although results of l-CT analysis look very

similar to histological and AAS scoring, there are

some peculiarities in what is measured and in what

defines the detection limits.

The calcified fraction of the aorta and its average

density cannot be determined by any other method than

l-CT. Histomorphometry only measures calcium

volume in a limited number of sections and hence a

significant error can be introduced due to sampling

bias, especially in case of a very limited calcification

level. A direct comparison remains impossible because

animal focused l-CT analysis can be completely

isotropic in contrast to histomorphometry that always

analyzes a restricted part of the sample volume.

AAS being the most accurate method of calcium

density measurement per wet weight is limited to an

integral value. As a result, there is no difference for

AAS whether calcium was concentrated in some

dense spots or uniformly distributed in the prepara-

tion. However, this latter information can be essential

for the investigator.

Another critical drawback of AAS is that, due to

its destructive character, this technique does not

allow comparing the results to histomorphometry

directly. It is not possible to perform AAS on fixed

tissue, since tissue processing may influence the

calcium content in the sample.

Previous feasibility studies of human coronary

artery wall in autopsy specimens [18] have shown that

in addition to the quantification of ectopic calcifica-

tions in the present report, l-CT can be successfully

applied in the field of atherosclerosis for plaques

visualization and coronary wall analysis. Again, a full

3D isotropic dataset provides an advantage of finding

and visualizing the most important spots and regions

that might be missed during histological sectioning.

The problem of in vivo observations of the same

plaque is complicated by the insufficient contrast of

clinical CT. To enhance soft tissue contrast a dual-

energy CT is emerging as a novel clinical instrument

[19, 20]. In this case CT utilizes the fact that relative

absorption of different elements is changing dramat-

ically as a function of X-ray photon energy. As a

future development l-CT may use the same principle

Int J Cardiovasc Imaging (2009) 25:615–624 623

123

for laboratory animal studies although the limitation

of lower signal-to-noise ratio compared to medical

CT remains valid.

Conclusions

In conclusion, l-CT proves to be a promising non-

invasive imaging technique allowing quantification of

ectopic calcification both in living animals and in

extracted tissues. This study is the detailed compar-

ison between l-CT (in vivo as well as ex vivo) and

traditional methods such as histomorphometry and

AAS.

In vivo investigations allow selecting ‘‘positive’’

animals from those that did not calcify when the

calcification level was higher than 5 mg/g of wet

tissue. In vivo l-CT was successful in 70% of the

animals.

l-CT analysis on prepared tissues proved to be

accurate to quantify aorta calcification with more that

95% reliability, providing a complete analysis of both

degree and distribution of the calcification while

remaining much less time consuming.

References

1. Davis GR, Wong FSL (1996) X-ray microtomography of

bones and teeth. Physiol Meas 17:121–146. doi:

10.1088/0967-3334/17/3/001

2. Paulus MJ, Gleason SS, Kennel SJ, Hunsicker PR, Johnson

DK (2000) High resolution X-ray computed tomography :

an emerging tool for small animal cancer research. Neo-

plasia 2:62–70. doi:10.1038/sj.neo.7900069

3. Ritman EL (2002) Molecular imaging in small animals:

roles for l-CT. J Cell Biochem 39(Supp):116–124. doi:

10.1002/jcb.10415

4. Namati E, Chon D, Thiesse J, Hoffman EA, de Ryk J, Ross

A, McLennan G (2006) In vivo l-CT lung imaging via a

computer-controlled intermittent iso-pressure hold (II-

BBH) technique. Phys Med Biol 51(23):6061–6075. doi:

10.1088/0031-9155/51/23/008

5. Marechal M, Luyten F, Nijs J, Postnov A, Schepers E, van

Steenberghe D (2006) Histomorphometry and micro-

computed tomography of bone augmentation under a tita-

nium membrane. Clin Oral Implants Res 16(6):708–714.

doi:10.1111/j.1600-0501.2005.01205.x

6. De Clerck NM, Meurrens K, Weiler H, Van Dyck D,

Vanhoutte G, Terpstra P, Postnov AA (2004) High reso-

lution X-ray microtomography for the detection of lung

tumors in living mice. Neoplasia 6:374–379. doi:10.1593/

neo.03481

7. Postnov A, Meurrens K, Weiler H, Van Dyck D, Xu H, Terpstra

P, De Clerck NM (2005) In vivo assessment of emphysema in

mice by high resolution X-ray microtomography. J Microsc

220:70–75. doi:10.1111/j.1365-2818.2005.01510.x

8. Waarsing JH, Day JS, van der Linden JC, Ederveen AG,

Spanjers C, De Clerck N, Sasov A, Weinans H (2004)

Detecting and tracking local changes in the tibiae of individual

rats : a novel method to analyse longitudinal in vivo l-CT data.

Bone 34(1):163–169. doi:10.1016/j.bone.2003.08.012

9. Postnov A, Zarowski A, De Clerck NM, Vanpoucke F, Offe-

ciers W, Van Dyck D, Peeters S (2006) High Resolution M-CT

scanning as innovatory tool for evaluation of the surgical

positioning of cochlear implant electrodes. Acta Otolaryngol

126(5):467–474. doi:10.1080/00016480500437377

10. Nair MK, Nair UP (2007) Digital and advanced imaging in

endodontics: a review. J Endod 33(1):1–6. doi:10.1016/j.

joen.2006.08.013

11. Persy V, Postnov A, Neven E, Dams G, De Broe M,

DHaese P, De Clerck NM (2006) Detection of vascular

calcifications by high resolution X-ray microtomography in

living rats with chronic renal failure. Arterioscler Thromb

Vasc Biol 26:2110–2116

12. De Clerck N, Postnov A (2007) High resolution X-ray

microtomography: applications in biomedical research. In:

Tavitian B, Leroy-Willig A, Ntziachristos V (eds) Inter-

national textbook of in vivo imaging in vertebrate. Wiley

& Sons, London, pp 57–77

13. Katsumata K, Kusano K, Hirata M, Tsunemi K, Nagano N,

Burke SK, Fukushima N (2003) Sevelamer hydrochloride

prevents ectopic calcification and renal osteodystrophy in

chronic renal failure rats. Kidney Int 64(2):441–450

14. Neven E, Dauwe S, De Broe ME, D’Haese PC, Persy V

(2007) Endochondral bone formation is involved in media

calcification in rats and in men. Kidney Int 72(5):574–581

15. Price PA, Roublick AM, Williamson MK (2006) Artery

calcification in uremic rats is increased by a low protein

diet and prevented by treatment with ibandronate. Kidney

Int 70:1577–1583

16. Feldkamp LA, Davis LC, Kress JW (1984) Practical cone-

beam algorithm. J Opt Soc Am A 1:612–619. doi:

10.1364/JOSAA.1.000612

17. Postnov A, Vinogradov A, Van Dyck D, Saveliev SV, De

Clerck NM (2003) Quantitative analysis of bone mineral

content by X-ray microtomography. Physiol Meas 24:165–

178. doi:10.1088/0967-3334/24/1/312

18. Langheinrich A, Bohle R, Greschus S, Hackstein G, von

Gerlach S, Rau W, Hoelschermann H (2004) Ahteroscle-

rotic lesions at micro-CT: feasibility for analysis of

coronary artery wall in autopsy specimens. Radiology

231:675–681. doi:10.1148/radiol.2313021718

19. Barreto M, Schoenhagen P, Nair A, Amatangelo S, Milite

M, Obuchowski N, Lieber M, Halliburton S (2008)

Potential of dual-energy computed tomography to charac-

terize atherosclerotic plaque: ex vivo assessment of human

coronary arteries in comparison to histology. J cardiovasc

comput Tomogr 2:234–242

20. Schwarz F, Ruzzics B, Schoepf J, Bastarrika G, Chiarmida S,

Abro J, Brothers Rm, Vogt S, Schmidt B, Costello P, Zwerner P

(2008) Dual-energy CT of the heart—principles and protocols.

Eur J Radiol 68:423–433. doi:10.1016/j.ejrad.2008.09.010

624 Int J Cardiovasc Imaging (2009) 25:615–624

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