Outcome of experimental stroke in C57Bl/6 and Sv/129 mice assessed by multimodal ultra-high field MRI.

RESEARCH Open Access
Outcome of experimental stroke in C57Bl/6 and
Sv/129 mice assessed by multimodal ultra-high
field MRI
Mirko Pham1,2*, Xavier Helluy3, Stefan Braeuninger4, Peter Jakob3, Guido Stoll4, Christoph Kleinschnitz4,
Martin Bendszus1,2
Abstract
Transgenic mice bred on C57Bl/6 or Sv/129 genetic background are frequently used in stroke research. It is well
established that variations in cerebrovascular anatomy and hemodynamics can influence stroke outcome in differ-
ent inbred mouse lines. We compared stroke development in C57Bl/6 and Sv/129 mice in the widely used model
of transient middle cerebral artery occlusion (tMCAO) by multimodal ultra-high field magnetic resonance imaging
(MRI).
C57Bl/6 and Sv/129 mice underwent 60 min of tMCAO and were analyzed by MRI 2 h and 24 h afterwards. Struc-
tural and functional images were registered to a standard anatomical template. Probability maps of infarction were
rendered by automated segmentation from quantitative T2-relaxometric images. Whole-brain segmentation of
infarction was accomplished manually on high-resolution T2-weighted (T2-w) RARE images. Cerebral perfusion
(cerebral blood flow, CBF) was measured quantitatively by modified continuous arterial-spin-labeling (CASL) and
apparent diffusion coefficients (ADC) by spin-echo diffusion-weighted imaging (DWI).
Probabilities of cortical (95.1% ± 3.1 vs. 92.1% ± 2.5; p > 0.05) and subcortical (100% vs. 100%; p > 0.05) infarctions
at 24 h were similar in both groups as was the whole-brain volumetric extent of cerebral infarction. In addition,
CBF and ADC values did not differ between C57Bl/6 and Sv/129 mice at any time point or region of interest.
The C57Bl/6 and Sv/129 genetic background is no major confounding factor of infarct size and cerebral perfusion
in the tMCAO model.
Introduction
The implementation of transgenic mice has also revolutio-
nized the field of experimental stroke research in that the
effects of distinct genes on stroke outcome can be easily
assessed. Most of transgenic mice originate from C57Bl/6
or Sv/129 inbred strains. Moreover, these strains are also
commonly used as “wild-type controls”. However, there
are considerable strain-related differences in the suscept-
ibility to cerebral ischemia. Variations in cerebrovascular
anatomy and hemodynamics as well as sensitivity to exci-
totoxicity have been identified as underlying reasons [1-7].
This may become of particular relevance when transgenic
mice with mixed genetic background are compared to
purebred controls. C57Bl/6 mice have been shown to be
more susceptible to ischemic injury compared to Sv/129
mice in models of global (forebrain) ischemia [8] and to
develop larger brain infarctions in permanent middle cere-
bral artery occlusion (pMCAO) [2,9]. Whether these find-
ings also extend to the most frequently used murine
stroke model, i.e. transient middle cerebral artery occlu-
sion (tMCAO) with an intraluminal thread, is not known.
Furthermore, important functional parameters of acute
ischemic brain damage such as cerebral perfusion or cyto-
toxic edema formation have not been systematically com-
pared between these two popular strains until now.
We recently introduced multimodal magnetic reso-
nance imaging (MRI) at high-field strength of 17.6 Tesla
as a powerful tool to non-invasively analyze the early
phase of cerebral ischemia and the evolution of infarc-
tions in individual mice over time [10]. In the present
study, C57Bl/6 and Sv/129 mice were subjected to 60
* Correspondence: mirko.pham@med.uni-heidelberg.de
1Department of Neuroradiology, University of Heidelberg, Im Neuenheimer
Feld 400, D-69120 Heidelberg, Germany
Pham et al. Experimental & Translational Stroke Medicine 2010, 2:6
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© 2010 Pham et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
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min tMCAO. MRI outcome measures were chosen to
characterize cerebral perfusion by continuous arterial
spin labeling (CASL), hypoxic diffusion restriction by
diffusion-weighted imaging (DWI), and irreversible
infarction by lesion extent on T2-w and quantitative T2
relaxometric images.
Methods
Experimental design and animal stroke model
All procedures and animal studies were approved by the
appropriate local authorities (Regierung von Unterfran-
ken, Wuerzburg, Germany) and conducted in accor-
dance with recommendations for the performance of
basic experimental stroke studies [11]. The experimental
groups consisted of 8 6-8-weeks-old male C57Bl/6 mice
(Charles River, Sulzfeld, Germany) and 8 6-8-weeks-old
male Sv/129 mice (Harlan-Winkelmann, Borchen,
Germany) weighing 20-25 g. Animals of each group
underwent tMCAO with 60 min occlusion time.
tMCAO was performed as described in detail pre-
viously [12-14]. Briefly, a standardized suture coated
with silicon rubber (6021PK10; Doccol Corporation,
Redlands, CA, USA) was introduced into the right com-
mon carotid artery and advanced via the internal carotid
artery to the origin of the middle cerebral artery (MCA).
The suture was fixed and left in situ and animals were
allowed to recover. Operation time per animal did not
exceed 15 min. After 60 min, animals were re-anesthe-
tized and the suture was withdrawn to allow tissue
reperfusion (tMCAO). The operations were performed
under inhalation anesthesia (2.0% isoflurane in a 70%/
30% N2O/O2 mixture) and the body temperature was
maintained at 37°C using a servo-controlled heating
pad. All animals were subsequently followed in-vivo by
serial multimodal ultra-high field MRI at 2 h and 24 h.
Multimodal ultra-high field MRI of experimental cerebral
ischemia in-vivo
The detailed description of the imaging protocol can be
found elsewhere [10]. A short summary of relevant para-
meters of the employed pulse sequences is given here.
Cerebral perfusion was measured using a modified arter-
ial spin labeling (CASL) method [15-17]. To benefit
especially from increased longitudinal magnetization and
the elevation of the T1 relaxation time for detailed ana-
tomical mapping of CBF and group analysis, all mea-
surements were performed at ultra-high field strength
(17.6 T, 750 Hz, Biospin, Bruker BioSpin GmbH, Ettlin-
gen, Germany). Image maps of cerebral perfusion were
calculated on a pixel-by-pixel basis according to Detre
et al. [15]. The degree of the inversion efficiency was
assumed to be alpha = 0.7 [18,19], and the brain-blood
partition coefficient value for water lambda = 0.95 mL/g
[20]. Parameters for the fast spin-echo imaging sequence
(RARE) were: echo train length (ETL) = 8, effective echo
time TEeff = 17.2 ms, repetition time TR = 1 s, slice
thickness 1.5 cm, FOV 2.5 × 2.5 cm, matrix of 64 × 64
voxels. The signal was averaged over 12 repetitions
resulting in a total acquisition time of 9.5 min.
DWI was performed with a pulsed-field gradient Setjs-
kal-Tanner-like multislice spin echo sequence with dif-
fusion sensitization along the slice direction [21]. Images
with different b-values, 0 and 800 s/mm2, were acquired
to allow for the calculation of apparent diffusion coeffi-
cient (ADC) maps of brain water. Whole brain coverage
was achieved by thirteen coronal slices acquired with a
matrix size of 64 × 64, FOV 2.5 × 2.5 cm, in plane reso-
lution 282 × 282 μm, slice thickness = 0.5 mm, inter-
slice distance = 1 mm, TE/TR = 22.3/2000 ms. Repeated
measurement of the b = 800 s/mm2 DWI experiments
(number of repetition NR = 3) resulted at an overall
acquisition time for diffusion weighted experiments of 8
min. ADC maps were calculated by applying the com-
mon equation ADC = -0.00125 × ln (SIB800/SIB0).
T2 relaxometric mapping was performed for the in-vivo
delineation of infarcted brain tissue at 24 h. Single slice T2-
w imaging was performed using a Carr-Purcell-Meiboom-
Gill (CPMG) multi-spin echo sequence collecting 32 echoes
at TR/TE = 4.2/2000 ms. T2 relaxation times constants
were calculated voxel-wise by fitting the intensities of the
20 first echoes to a monoexponential model. In addition, a
strongly T2 weighted 2D turbo spin-echo sequence was
acquired for high resolution anatomical imaging (RARE
factor 16, TR = 8 s, effective TE = 56.44 ms, 2 averages, 13
coronal slices with an image matrix of 128 × 128 were
acquired, FOV = 2.5 cm × 2.5 cm, slice thickness =
0.5 mm, interslice distance = 1 mm, overall acquisition
time of 1 min). The center position of the 1.5 mm slab for
measurement of CBF, T1 and T2 relaxometric maps each
with the exact same geometry was centered at the bregma
as the operational definition of the central MCA territory.
At the host console measurements and data proces-
sing were performed with the ParaVision software
(version 3.02, Bruker BioSpin GmbH, Ettlingen,
Germany). Further image calculation and fitting proce-
dures were done using MATLAB® (The Mathworks Inc.,
Natick, MA, USA).
During MRI measurements, mice were anesthesized by
2% isoflurane in medical air (21%). The respiratory rate
was monitored using an air-balloon positioned ventrally
underneath the mouse body. The body temperature was
constantly measured on the body surface and actively
maintained at 37°C.
Statistical and image analysis
The extraction of brain tissue from the scalp and skull
was done by manual segmentation for each subject
and time point. Packages from the FMRIB Software
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Library FSL (version 4.1) [22] were used for motion
correction, registration (FLIRT) [23], and statistical
image analysis. Intra-subject linear alignment and
registration to a common standard template [24] was
achieved by a step-wise affine procedure with six
degrees of freedom.
For quantitative group comparisons, selected regions
of interest (ROIs) were delineated in atlas space: 1) the
cerebral cortex in the center of the MCA territory and
2) the subcortex including the ipsilateral caudoputamen
and pyramidal tract (Figure 1). Statistical analysis of
ROIs was done by non-parametric pairwise comparisons
using the Wilcoxon matched-pairs signed-rank test. The
risk of cerebral infarction was determined on within-
group probability maps calculated by averaging the bin-
ary segmentations of healthy versus infarcted brain tis-
sue within-subject. Automated binary segmentation of
cerebral infarction was performed by applying a thresh-
old of 34 ms T2 relaxation time on the T2 relaxometric
maps at 24 h as demonstrated previously [10]. Manual
input was given only for the removal of intraventricular
cerebrospinal fluid. Finally, whole-brain volumetric ana-
lysis of infarction was done by manual segmentation on
the images of the T2-w RARE sequence. Values are
always given as mean ± standard error of the mean
(SEM).
Results
Manual whole-brain segmentation of infarction
Volumetric extents of cerebral infarction as delineated
manually on T2-w RARE imaging are given as volume
ratios (infarction/hemisphere) and were as follows for the
C57Bl/6 mice: at 2 h 0.1 ± 0.03 and at 24 h 0.43 ± 0.02.
For the Sv/129 mice, values were 0.06 ± 0.03 at 2 h and
0.37 ± 0.03 at 24 h. No statistical differences were found
when comparing pairwise-matched groups (Figure 2).
Probability maps of infarction by quantitative T2
thresholds
Automated segmentation of infarction for each indivi-
dual animal was performed with a threshold of a quan-
titative T2 value of 34 ms as described previously [10].
The probabilities of infarction in atlas space are given
in Table 1 for both groups, time points and corre-
sponding subcortical and cortical ROIs. There was no
significant difference in the probability of infarction
between both groups at 2 h (p = 0.4) or 24 h (p =
0.93). The spatial distribution of the different probabil-
ities of infarction is demonstrated on color-coded
probability maps and relaxometric T2 maps (Figure 3).
Interestingly, subcortical infarctions (basal ganglia)
were present already 2 h after stroke onset correspond-
ing to the insufficient collateral blood supply in these
Figure 1 Region-of-interest masks. On the left the central cortical territory of the middle cerebral artery (MCA) is delineated by the white
overlay region according to the corresponding coordinates in atlas space, and the deep subcortical territory of the MCA is displayed on the
right.
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areas. After 24 h infarction has extended to the whole
MCA territory including the cortex.
Analysis of functional ischemic outcome parameters
Pairwise comparisons between group means of quantita-
tive CBF values were not significant for any time point or
region of interest. Two-sided p-values for matched pair-
wise comparisons between C57BL/6 and Sv/129 mice
were: subcortical ROI at 2 h p = 0.7 and p = 0.53 at 24
h; cortical ROI at 2 h p = 0.64 and p = 0.86 at 24 h.
Similarly, ADC values in the same ROIs showed no sta-
tistical differences between group means. Two-sided p-
values for matched pairwise comparisons between C57BL/
6 and Sv/129 mice were: subcortical ROI at 2 h p = 0.34
and at 24 h p = 0.91; cortical ROI at 2 h p = 0.28 and at
24 h p = 0.63. Quantitative mean values for CBF and ADC
are given in Table 2. Figure 4 displays the spatial distribu-
tion of functional outcome measures on color maps.
Again, infarctions in both groups evolved from the subcor-
tical to the cortical areas over time.
Discussion
As principle finding, we here show that C57Bl/6 and Sv/
129 mice, the two most frequently utilized strains in
experimental stroke research, behave similar in the
tMCAO model regarding critical functional and struc-
tural parameters of infarct development. Volumetric
extents and probability of cerebral infarctions did not
significantly differ between the two groups as assessed
by multimodal ultra-high field MRI as did cerebral per-
fusion and diffusion restriction of free water.
Figure 2 Group means of infarct volume ratios (infarction/hemisphere). Pairwise comparisons between 8 C57Bl/6 and Sv/129 mice did not
reveal any statistical differences for the 2 h (p = 0.204) or 24 h time point (p = 0.172) after tMCAO. Error bars denote standard errors of the
mean.
Table 1 Group means of infarct probabilities (%).
129/Sv C57BL/6
2 h 24 h 2 h 24 h
Cortex 53.3 ± 10.1 92.1 ± 2.5 60.7 ± 10.4 95.1 ± 3.1
Subcortex 67.8 ± 12.8 100 79.1 ± 11.3 100
Values are given for both cortical and subcortical ROIs and both time points
after tMCAo. Results of statistical pairwise comparisons are given in the text
and did not significantly differ between groups for any time point. Standard
errors of the mean are denoted by ±.
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Strain-related differences between C57Bl/6 and Sv/129
mice have already been investigated in models of global
and permanent focal cerebral ischemia. C57Bl/6 mice
challenged by transient bilateral common carotid artery
occlusion in the presence or absence of systemic hypo-
tension developed more severe global forebrain ischemia
than Sv/129 mice [7,8]. Results after permanent MCAO
have been inconclusive. While some reports described
larger infarctions in C57Bl/6 mice in direct comparison
to the Sv/129 strain [2,9], others could not confirm this
observation [4]. We further extend these findings to the
most frequently used model of focal cerebral ischemia,
i.e. tMCAO with an intaluminal thread. Here, 60 min of
focal ischemia had no differential effect on definite
infarct volumes on day 1. Importantly, diffusion restric-
tion of free water as an early marker of ischemic cell
damage and cytoxic edema likewise occurred similar in
both strains. This implies that the C57Bl/6 or Sv/129
genetic background is no major confounding factor of
infarct evolution in the acute phase after tMCAO.
Whether this also applies to later stages of infarct devel-
opment or different MCA occlusion times, however, still
needs to be assessed.
Besides infarct volume, regional cerebral blood flow
(rCBF) is frequently chosen as readout parameter in
experimental stroke studies. Both critically depend on
the composition of the cerebral vasculature which is
known to vary considerably between different mouse
strains [1-3]. The posterior communicating artery is
absent or hypoplastic in more than 90% of C57/Bl6
mice, but in less than 50% of Sv/129 mice, which is
also supported by in-vivo MRI data [4,25]. Moreover,
Figure 3 Color coded group means of probability of infarction and quantitative T2 values. For both groups probability of cortical and
subcortical infarction was similar. Note that subcortical infarction is evident on T2 maps already 2 h after tMCAO. The segmentation threshold
for infarction was set to 34 ms.
Table 2 Group means of functional ischemic outcome measures: CBF and ADC.
Sv/129 C57Bl/6
CBF
(ml/100g/min)
2 h vs. 24 h
Cortex 34.1 ± 4.7 vs. 22.3 ± 2.9 38.7 ± 4.8 vs. 25.2 ± 3.7
Subcortex 30.4 ± 4.5 vs.20.8 ± 2.7 32.8 ± 4.6 vs. 23.4 ± 3.5
ADC
(mm2/s*10-4)
2 h vs. 24 h
Cortex 7.8 ± 1.0 vs. 5.8 ± 0.5 7.8 ± 1.0 vs. 5.7 ± 0.6
Subcortex 7.3 ± 1.0 vs. 5.4 ± 0.5 7.2 ± 1.1 vs. 5.3 ± 0.4
Values are given for both cortical and subcortical ROIs and compared between both time points. P-values of statistical comparisons for CBF and ADC between
groups were not significant and are given in the text. Standard errors of the mean are denoted by ±.
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