Inadvertent Occlusion of the Anterior Choroidal Artery Explains Infarct Variability in the Middle Cerebral Artery Thread Occlusion Stroke Model
et al. (2013) Inadvertent Occlusion of the Anterior Choroidal Artery Explains Infarct Variability in
the Middle Cerebral Artery Thread Occlusion Stroke Model. PLoS ONE 8(9): e75779. doi:10.1371/journal.pone.0075779
Inadvertent Occlusion of the Anterior Choroidal Artery Explains Infarct Variability in the Middle Cerebral Artery Thread Occlusion Stroke Model
Neil J. Spratt 0
Damian D. McLeod 0
Daniel J. Beard 0
Mark W. Parsons 0
Christopher R. Levi 0
Mike B. Calford 0
Thiruma V. Arumugam, National University of Singapore, Singapore
0 1 University of Newcastle and Hunter Medical Research Institute , Callaghan, New South Wales , Australia , 2 Hunter New England Local Health District, New Lambton Heights , New South Wales , Australia
Intraluminal occlusion of the middle cerebral artery (MCAo) in rodents is perhaps the most widely used model of stroke, however variability of infarct volume and the ramifications of this on sample sizes remains a problem, particularly for preclinical testing of potential therapeutics. Our data and that of others, has shown a dichotomous distribution of infarct volumes for which there had previously been no clear explanation. When studying perfusion computed tomography cerebral blood volume (CBV) maps obtained during intraluminal MCAo in rats, we observed inadvertent occlusion of the anterior choroidal artery (AChAo) in a subset of animals. We hypothesized that the combined occlusion of the MCA and AChA may be a predictor of larger infarct volume following stroke. Thus, we aimed to determine the correlation between AChAo and final infarct volume in rats with either temporary or permanent MCA occlusion (1 h, 2 h, or permanent MCAo). Outbred Wistar rats (n = 28) were imaged prior to and immediately following temporary or permanent middle cerebral artery occlusion. Presence of AChAo on CBV maps was shown to be a strong independent predictor of 24 h infarct volume (b = 0.732, p ,0.001). This provides an explanation for the previously observed dichotomous distribution of infarct volumes. Interestingly, cortical infarct volumes were also larger in rats with AChAo, although the artery does not supply cortex. This suggests an important role for perfusion of the MCA territory beyond the proximal occlusion through AChA-MCA anastomotic collateral vessels in animals with a patent AChAo. Identification of combined MCAo and AChAo will allow other investigators to tailor their stroke model to reduce variability in infarct volumes, improve statistical power and reduce sample sizes in preclinical stroke research.
Funding: This work was supported by the National Health and Medical Research Council (NHMRC, Australia), Program Grant, #454417 (www.nhmrc.gov.au); the
National Stroke Foundation (Australia), Small Project Grant (http://strokefoundation.com.au/research/research-grants-2008-09/); the Hunter Medical Research
Institute Stroke Research Project Grant (Grant #G0189810) (www.newcastle.edu.au); and from funds donated by the Greater Building Society (http://www.greater.
com.au). N. Spratt received a NHMRC training fellowship, #455632 (www.nhmrc.gov.au). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interest exist.
Studies in both human and animals have reported large
variability in the vascular anatomy of the brain. Of significance
in the field of ischemic stroke research is the anatomy of the
middle cerebral artery (MCA) and adjacent branches, in particular
the anterior choroidal artery (AChA). The AChA supplies blood to
the globus pallidus, the internal capsule, choroid plexus and has
anastomotic connections with the MCA in rats and humans [1,2].
Despite the AChA territory being well defined, there is substantial
variability in the origin of this vessel. The AChA predominantly
originates from the internal carotid artery (ICA) in close proximity
to the MCA in rats and humans .
Intraluminal thread occlusion of the MCA in the rat is the most
common experimental model of stroke. This technique is
performed by inserting an occluding monofilament suture via
the ICA to occlude the origin of the MCA . Advantages of this
technique include lack of craniotomy and control over occlusion
and reperfusion. One disadvantage of this model is the large
variability in infarct volume. Factors thought to influence infarct
volume variability include genetics , blood pressure ,
collateral blood flow , duration of vessel occlusion, and type
of intraluminal thread used .
A new observation was made while developing our perfusion
computed tomography (CTP) rat stroke model (intraluminal
MCAo) [9,10]. In a subset of animals, there was loss of
arterialintensity signal in the distribution of the anterior choroidal artery
(AChA) on cerebral blood volume (CBV) maps, in addition to the
expected loss of signal in the MCA territory. We hypothesized that
the combined occlusion of the MCA and AChA may be a
predictor of larger infarct volume at 24 h following stroke. Thus
we aimed to determine the correlation between AChAo and final
infarct volume, in rats with either temporary or permanent MCA
occlusion (1 h, 2 h, and permanent MCAo). The aims of the
project were successfully achieved.
All surgical and experimental protocols were in accordance with
the Australian Code of Practice for the Care and Use of Animals
for Scientific Purposes and were approved by the University of
Newcastle (UoN) Animal Care and Ethics Committee.
Male outbred Wistar rats (300500 g) (Animal Services Unit,
UoN) underwent MCAo, using the silicone-tipped intraluminal
thread occlusion method . Each 4-0 monofilament occluding
thread had a 3 mm silicone tip (0.35 mm diameter). Animals were
anesthetized with isoflurane (5% induction, 1.5 2.5%
maintenance during surgery and 1.01.5% during imaging) and a 50:50
mix of nitrous oxide and oxygen via facemask. Animals were
subject to either permanent MCAo (n = 13), or 1 hr (n = 8) or 2 hr
(n = 7) temporary MCA occlusion, with reperfusion achieved by
gentle withdrawal of the occluding thread. Details of thread
insertion for MCAo on the CT scanning table have been
previously reported . All animals had a jugular venous catheter
inserted for injection of radio-opaque contrast during imaging .
CT Image Acquisition, Processing and Analysis
Detailed methods and perfusion computed tomography (CTP)
data have been reported for this cohort of animals [9,10]. All
imaging was performed using a Siemens 64 slice CT scanner
(Siemens, Erlangen, Germany) with a 5126512 matrix, 50 mm
field of view (FOV) with twelve 2.4 mm slices.
Post-processing of perfusion data was performed using MIStar
imaging software (Apollo Medical Imaging Technology,
Melbourne, Australia), as previously described . In brief, cerebral
blood flow (CBF), cerebral blood volume (CBV), and mean transit
time (MTT) maps were generated from CTP images recorded
within 5 minutes after MCAo. The two CTP slices that were
analyzed were located at 0 mm bregma, and at 2.4 mm caudal to
bregma. This allowed assessment of perfusion changes in the
MCA and AChA territories. First, to exclude voxels containing
large blood vessels the standard CBV large vessel threshold
(10 ml/100 g) [9,10] was applied. This threshold did not exclude
voxels within the AChA territory, however this territory was
clearly visible on CBV colour maps. Therefore, a more stringent
threshold was used to provide an objective marker of presence or
absence of AChA occlusion. To do this, regions-of-interest (ROIs)
encompassing the left (contralateral) and right (ipsilateral)
hemispheres were generated (Figure 1). To determine the presence of
concurrent AChAo, a threshold greater than or equal to the mean
contralateral CBV was applied. Lack of signal in the ipsilateral
AChA territory on these maps indicated occlusion of the AChA.
Data analysis was performed by an observer (DDM) blinded to
infarct volume outcome.
At 24 hours following MCA occlusion, rats were euthanased by
transcardial perfusion fixation with 4% paraformaldehyde. This
was followed by histological processing, hemotoxylin-eosin (H&E)
staining, scanning at 20x objective on a digital slide scanner
(Aperio Technologies Inc., Vista, CA, USA), and infarct
quantification, as previously described . Infarct volumes were
corrected for edema using the formula: corrected infarct vol.
(mm3) = infarct vol. x (contralateral vol./ipsilateral vol.). Infarct
probability maps were generated for the pMCAo group at two
bregma levels (0.3 mm and 2.3 mm bregma) using Adobe
Photoshop Version 13.0 (Adobe Systems Incorporated). The
Photoshop Warp tool was used to transform the histological
outlines to fit the stereotaxic atlas template (Paxinos and Watson
). The traced infarct outlines were filled with the same grey
colour and the opacity was set to 24%. Sections from subsequent
animals were overlaid.
Statistical tests were performed with Graphpad Prism 6 (CA,
USA) and SPSS 21.0 (IBM, USA). Due to the retrospective nature
of the study, DAgostino & Pearson omnibus normality tests were
performed on all data prior to further statistical analyses. If data
was normally distributed (alpha . 0.05), two-tailed t-tests were
performed. The effect of MCAo vs. MCAo + AChAo on infarct
volume within each of the 1 h, 2 h, and pMCAo groups, and in all
animals combined were analyzed. All other statistical analyses
between groups were done with two-tailed t-tests.
Regression analysis was used to determine independent
predictors of infarct volume. The predictors of interest were
MCA occlusion duration, the patency of AChA (i.e. occluded or
not occluded), and weight. Each of these predictors was examined
with a Pearson correlation analysis, and those with p,0.10 were
included in a multiple linear regression model. The standardized
coefficients (b-value) and p-values are presented to indicate the
significant predictors of infarct volume. Statistical significance was
accepted at p,0.05. Data is presented as mean 6 SD.
Successful occlusion of the MCA was observed in all animals
(n = 28) by visual inspection of the CTP maps (Figure 1). Infarction
was confirmed in all animals with H&E histology at 24 h (Figure
2). A subset of animals (n = 10) had no CBV above the mean
contralateral CBV in the ipsilateral AChA territory, indicating
AChAo (Figure 1B).
Infarct volumes from the pMCAo and 1 h groups were normally
distributed (p = 0.261, p = 0.068, respectively). However, no further
statistical analyses were performed on the 1 and 2 h groups
because there were only two and one animal respectively in each
of these groups with MCAo + AChAo. There were significant
differences in infarct volume between animals with MCAo +
AChAo compared to MCAo alone in the pMCAo group
(42.7627.62 mm3 vs. 144.5611.72 mm3, p,0.0001, Figure 3A).
When infarct volume data from the 1 h, 2 h and pMCAo groups
were pooled into MCAo and MCAo + AChAo groups, there was a
significant difference in infarct volume (24.6623.38 mm3 vs.
129.6641.58 mm3, p,0.0001).
AChAo patency and MCAo duration were significantly
correlated with infarct volume (r = 0.85, p,0.0001; r = 0.58,
p = 0.0013, respectively). There was no significant correlation
between weight and infarct volume (r = 0.22, p.0.10). AChAo
patency and MCAo duration were included in the subsequent
multiple linear regression model, which had an adjusted r2 value of
0.831. AChA patency and occlusion duration were both found to
be significant independent predictors of 24 h infarct volume with
b = 0.732 and 0.367 respectively (both p,0.001).
Interestingly, the increase of infarct volume in the MCAo +
AChAo group was not restricted to the territory of the AChA,
which is entirely subcortical, but was also seen in cortical regions
(Figure 2). Quantification of cortical and sub-cortical volumes of
infarction in the pMCAo group revealed significantly larger
subcortical infarct volumes (59.369.11 mm3 vs.
28.5615.93 mm3, p = 0.001), and even more dramatic differences
in cortical infarct volumes in the MCAo + AChAo animals
compared to those with MCAo alone (85.269.50 mm3 vs.
14.2616.98 mm3, p,0.00001; Figure 3B & C).
In this study, our goal was to determine the importance of
inadvertent AChAo to the major problem of infarct volume
variability in the intraluminal thread occlusion model. We showed
that it had a major effect. Presence of anterior choroidal artery
occlusion on CTP-derived CBV maps was a strong independent
predictor of final infarct volume. The relationship between
AChAo and infarct volume was present with either permanent
or temporary occlusion. Although the known relationship between
MCAo duration and infarct volume was also observed, within
each occlusion duration group animals with AChAo had larger
infarcts than those without. The use of CTP to define anterior
choroidal artery occlusion has provided the likely explanation for
the dichotomous distribution of infarct volumes after intraluminal
thread occlusion of the MCA in rats [11,13,14]_ENREF_11.
Occluding filament design, variability of vascular anatomy, and
occluder tip positioning are likely causes for variability of AChAo.
In the current study, variable occlusion of the AChA during
MCAo occurred using occluding tips with consistent silicone tip
Figure 2. Histology sections from two pMCAo rats 24 h after stroke. Hemotoxylin-eosin stained coronal sections scanned at 20x objective.
The animal with the MCAo + AChAo (right column) had prominent subcortical and cortical infarction (pyknotic, dark, shrunken nuclei: expanded
regions-of-interest); the animal with MCAo alone (left column) had subcortical infarction, but no infarction within similar cortical locations.
Figure 3. Effect of concurrent anterior choroidal artery occlusion on infarct volume after temporary or permanent MCA occlusion.
Outbred Wistar rats underwent 1 h (n = 8), 2 h (n = 7) or permanent MCAo (n = 13) of the right middle cerebral artery (MCAo) using a silicone-tipped
intraluminal filament. A. Infarct volume was quantified with histology at 24 h. Presence of anterior choroidal artery occlusion (AChAo) was determined
by cerebral blood volume (CBV) map analysis. B. Cortical and sub-cortical infarct volume distribution in pMCAo animals (mean + sd). C. The traced
infarct (seen in grey) from animals in the pMCAo group were overlaid so that lighter regions represent areas more commonly infarcted.
length and diameter (3 mm60.35 mm, respectively). Previous
studies have shown that occluder tips with larger diameters produce
larger infarct volumes [8,15,16]. Variability of infarct volumes
with different tip lengths has also been investigated . Combined
MCAo and AChAo was reported to occur with occluder tip
lengths .3.3 mm, but not with shorter tip lengths . However,
studies have also shown that the distance between the AChA and
MCA is variable within and across rat strains [3,17]. Another
important variable may be the final positioning of the occluding
thread. Ma et al (2006) demonstrated that the final position of
silicone-tipped occluders (with constant tip lengths), varied within
and between six different rat strains, .
An important question arising from this research is why AChA
occlusion is such an important determinant of infarct volume?
Infarction within the arterial territory of the AChA itself does not
appear to be a sufficient explanation for this phenomenon. In the
rat and human, the anterior choroidal artery typically supplies a
small proportion of the sub-cortex and choroid plexus [1,2].
Although sub-cortical infarction was significantly greater with
occlusion of the MCA and AChA, this does not explain why
cortical infarction was also significantly larger than MCA
occlusion alone. The likely explanation is that occlusion of the
AChA prevents collateral flow to the MCA territory via the known
MCA-AChA collateral anastomotic vessels .
In summary, the intermittent occurrence of inadvertent AChA
occlusion during filament occlusion of the MCA may be a major
contributor to infarct volume variability. Techniques such as CTP
can be used to determine the presence or absence of AChA
occlusion. We hope that this finding helps to alert researchers to
this important predictor of infarct volume and hence potential
cause of model variability. This may enable other laboratories to
tailor their model to reduce outcome variability, improve statistical
power and reduce sample sizes in preclinical stroke research.
We would like to thank Qing Yang for the design of the MIStar imaging
software (Apollo Medical Imaging Technology, Melbourne, Australia); the
Faculty of Health Stores Workshop of the University of Newcastle for
manufacture of CT compatible anesthetic equipment; Debbie Pepperall,
Sunyoung Chung and Dr. Sarah McCann for histopathology; and Marc
Heaton and David Buxton for radiography.
Conceived and designed the experiments: DDM MWP CRL MBC NJS.
Performed the experiments: DDM DJB MWP CRL NJS. Analyzed the
data: DDM DJB NJS. Contributed reagents/materials/analysis tools:
DDM DJB MWP CRL MBC NJS. Wrote the paper: DDM DJB MWP
CRL MBC NJS.
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