The simultaneous use of multiple imaging techniques to
investigate the nature and localisation of radioactivity within
micro-structures surrounding the rat brain
Karen Stevenson, Craig Mackay, Lyndsay Brogan - Charles River Edinburgh
Marie Molander-Melin, Bengt Larsson, Per Åberg, Philip Gardiner - AstraZeneca
EUROTOX, EDINBURGH
Quantitative whole body autoradiography
(QWBA) is a powerful and well documented
technique used to determine the relative tissue
distribution of radiolabelled drugs. It is the tool
of choice for investigating drug-related
distribution to target and non-target organs in
preclinical species. In conjunction with QWBA,
micro-autoradiography (MARG) is increasingly
being used to pinpoint the localisation of drugrelated material within tissues and microstructures at the cellular or sub-cellular level.
08/2014
In this study, QWBA, MARG and
histopathological evaluations (including
haematoxylin and eosin (H&E) analysis) were
combined to investigate the localisation of
radioactivity within micro-structures surrounding
the rat brain. Due to the complex nature of the
brain and surrounding structures this
investigation used the coronal and transverse
planes as well as the traditional sagittal plane,
thus providing a “mapping” of drug-related
radioactivity within these structures.
Each technique provided unique information.
QWBA enabled the determination of the
concentration of total radioactivity within the
structures of interest, however QWBA could not
localise the radioactivity within the microstructures. MARG analysis allowed for this
localisation to be determined, and using these
images in combination with slides prepared for
histopathological evaluation a qualitative
analysis of cell or structural location was
possible. The preparation of the slides for MARG
and H&E analysis using equipment designed for
macro QWBA was technically very challenging
and how this was overcome is described. The
study demonstrates that the application of these
techniques can provide useful information which
due to the nature and positioning of the
structures of interest would not have otherwise
been possible.
•A further 1 pigmented and 1 albino male rat
euthanised at 168 h post dose.
• Each rat immediately frozen by immersion
in a mixture of solid CO2 and hexane for ca
30 min.
•The head of each animal removed and
embedded in methylcellulose, which was
frozen using the same dry ice/hexane
mixture.
•At each timepoint, 1 pigmented and 1 albino
rat was sectioned in the coronal and
transverse plane. At 168 h post dose, the
additional animals were sectioned in the
sagittal plane.
•Sections (30 µm) were taken through each
head using a Leica CM3600 Cryomicrotome
(Leica Instruments, Germany) and freeze
dried for autoradiography.
•An additional section (8-10 µm) was taken
and transferred onto a glass slide using the
Cryojane® tape transfer system (Leica
Biosystems, USA ). These sections were
stained with haematoxylin and eosin.
•Sections for H&E staining were completed
on Autostainer XL (Leica Microsystems,
Germany) and then cover slipped (Leica
CV5030, Leica Microsystems, Germany).
•At 1 selected level additional sections
(8 µm) were taken via the tape transfer
method and processed for MARG analysis.
Results
A selection of images produced from the
three types of analysis are shown.
Introduction
In this investigation a novel test item was
radiolabelled with carbon-14 in a metabolically
stable position on the structure of the molecule.
The [14C]-test item was administered orally to
both pigmented (Lister Hooded) and albino (Han
Wistar) male rats. The distribution of
radioactivity in the micro-structures surrounding
the brain was determined using QWBA.
Additional sections were taken for MARG and
H&E analysis in order to allow localisation of the
radioactivity within the micro-structures of
interest.
Materials and Methods
•Oral dose formulation containing [14C]-test item
was prepared at Charles River Preclinical
Services, Edinburgh.
•Rats were obtained from Charles River (UK)
Limited.
•All regulated procedures carried out on the
animals were performed under licences
authorised by the terms and conditions of the UK
Animals (Scientific Procedures) Act 1986.
•Radioactive dose of 5 MBq/kg.
•Two pigmented and 2 albino male rats were
humanely euthanised by CO2 narcosis at each of
24, 168 and 672 h post dose.
Figure 1: Autoradiogram
image from a pigmented
animal showing distribution
of radioactivity
Figure 3: H&E image
showing melanin pigments
present in same location as
radioactivity
Figure 2: MARG image
showing silver grain
deposition in corresponding
regions
Figure 4: representative
coronal autoradiogram
image from a pigmented
animal
Figure 7: Sagittal autoradiogram
from albino animal showing no
radioactivity present around the
brain
Figure 8: lack of silver
grain deposition
consistent with no
presence of radioactivity
Figure 9: H&E image from
an albino animal showing
no melanin pigments
present
Experimental Adaptations
This was the first time the CM3600 had been
used for obtaining sections for MARG and H&E
analysis.
Unlike the sections used for autoradiography,
which are taken directly onto pressure sensitive
tape (Filmolux 610 tape, Neschen AG,
Germany), sections for H&E and MARG analysis
were required to be transferred to glass slides
for analysis. The most effective method of
conducting this was to use the CryoJane® tape
transfer system.
Due to the location of the microstructures of
interest it was imperative to maintain the integrity
of the skull, brain and surrounding tissues during
sectioning. Normally a Cryotome (Thermo
Scientific, Cheshire, UK) would be used for
sectioning frozen tissue for MARG typically
between 4-8 µm. However due to the difficultly of
sectioning through thick bone present in the
skull, it was beyond the Cryotome capabilities.
Formalin Fixed Paraffin Embedded (FFPE)
sections could also be used in MARG however
when sectioning on the Cryotome, the thick bone
would have to under go decalcification, which
would potentially cause translocation of the [14C]test item and disrupt the microstructures of
interest.
The CM3600 together with the CryoJane tape
transfer system obtained reasonable quality
frozen sections at 8 µm which required a great
deal of technical skill and ability. Through this
combination of techniques and expertise the
project was able to successfully identify and
locate the areas of interest.
Conclusion
Figure 5: MARG image
showing silver grain
deposition in meninges
corresponding to location of
radioactivity in
autoradiogram in Figure 4
Figure 6: H&E image
showing location of melanin
pigments consistent with
silver grain staining
Following Autoradiography, MARG and H&E
staining of selected sections from the heads of
pigmented and albino animals the radioactivity
distributed to tissue surrounding the brain in
pigmented rats was determined to be attributable
to melanin pigments located within the meninges
of the brain, specifically those around cranial
blood vessels.