Complications and Pitfalls in Rat Stroke Models for Middle
Cerebral Artery Occlusion
A Comparison Between the Suture and the Macrosphere Model Using
Magnetic Resonance Angiography
Tibo Gerriets, MD; Erwin Stolz, MD; Maureen Walberer, DVM; Clemens Müller, PhD;
Carina Rottger, MD; Alexander Kluge, MD; Manfred Kaps, MD;
Marc Fisher, MD; Georg Bachmann, MD
Downloaded from http://stroke.ahajournals.org/ by guest on June 18, 2017
Background and Purpose—Investigating focal cerebral ischemia requires animal models that are relevant to human stroke.
Complications and side effects are common among these models. The present study describes potential pitfalls in 3
techniques for middle cerebral artery occlusion (MCAO) in rats using magnetic resonance imaging (MRI) and magnetic
resonance angiography (MRA).
Methods—Rats were subjected to temporary MCAO for 90 minutes using the suture technique (group I; n⫽10) or to
permanent MCAO using the suture technique (group II; n⫽10) or the macrosphere technique (group III; n⫽10). Clinical
evaluation was performed after 3 hours and 24 hours. After 24 hours, animals underwent MRI and MRA to determine
lesion size and the intracranial vascular status.
Results—Hemispheric lesion volume was significantly smaller in group I (14.6%) compared with groups II (35.2%;
P⬍0.01) and III (21.3%; P⬍0.05). Two animals (1 each in group II and III) did not demonstrate neurological deficits
and had no lesion on MRI and a patent MCA main stem on MRA. Subarachnoid hemorrhage was detected in 2 animals
(1 each in group I and II). MRA indicated a patent MCA main stem in 2 animals (group II), although both rats displayed
neurological deficits. Hypothalamic infarction with subsequent pathological hyperthermia was detected in all animals
in group II and in 1 rat in group III.
Conclusions—Model failures occurred frequently in all groups. MRI and MRA helps to identify animals that need to be
excluded from experimental stroke studies. (Stroke. 2004;35:2372-2377.)
Key Words: hyperthermia 䡲 magnetic resonance angiography 䡲 magnetic resonance imaging 䡲 stroke
A
nimal models of focal cerebral ischemia allow study of stroke
pathophysiology and evaluation of new therapeutic approaches. Among different animal models available for focal
cerebral ischemia induction, endovascular techniques are less invasive and therefore preferable.1 Among the endovascular techniques
for middle cerebral artery occlusion (MCAO), the suture occlusion
technique in rats is the most frequently used method.2 In this model,
a monofilament is advanced into the internal carotid artery (ICA)
until it blocks blood flow to the middle cerebral artery (MCA). This
technique provides reproducible MCA territory infarctions and
allows reperfusion by retracting the suture. Permanent MCAO with
the suture technique, however, has one important disadvantage:
insertion of the suture occludes the entire course of the ICA, leading
to obstruction of the hypothalamic artery (HA). This causes hypothalamic infarction with associated pathologic hyperthermia that
confounds neuroprotective drug evaluation.3–5 The recently intro-
duced macrosphere model has been developed to overcome this
side effect by the intra-arterial embolization of 6 TiO2 spheres that
selectively block blood flow to the MCA main stem without
obstructing the HA. This model avoids hypothalamic infarction and
pathological hyperthermia but does not allow for reperfusion.4,5,6
The present study was designed to evaluate validity,
complications, and side effects of 3 different MCAO models
(permanent MCAO using the suture and the macrosphere
models and transient occlusion with the suture technique).
MRI and MRA have been applied to identify model failures.
Materials and Methods
Animal Preparation
All procedures are in accordance with our institutional guidelines
and the German animal protection legislation.
Received May 14, 2004; final revision received June 23, 2004; accepted July 26, 2004.
From the Department of Radiology (T.G., E.S., M.W., C.M., A.K., G.B.), Experimental Neurology Research Group, Kerckhoff-Klinik Bad Nauheim,
Germany; the Department of Neurology (T.G., E.S., M.W., C.R., M.K.), University Giessen, Germany; and the Department of Neurology (M.F.),
University of Massachusetts Medical School, Worcester, Mass.
Correspondence to Dr Georg Bachmann, Kerckhoff-Klinik Bad Nauheim, Department of Radiology/Experimental Neurology Research Group,
Benekestrasse 2-8, 61231 Bad Nauheim, Germany. E-mail [email protected]
© 2004 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000142134.37512.a7
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Gerriets et al
Thirty male Sprague-Dawley rats (290 to 350 grams; Harlan
Winkelmann, Borchen, Germany) were anesthetized with 5% isoflurane delivered in air for 2 minutes. Anesthesia was maintained with
2% to 3% isoflurane delivered in air at 0.5 L/min during surgery.
Body temperature was continuously monitored with a rectal probe
and maintained at 36.5°C to 37.0°C.
The right external carotid artery (ECA) was ligated and transsected to create an ECA stump with a length of ⬇5 mm. The
pterygopalatine branch of the ICA was also occluded.
Suture Models
A 4-0 silicone– coated nylon suture was introduced through the ECA
stump as described previously.4 The occluder was advanced into the
ICA 16 to 18 mm beyond the carotid bifurcation until mild resistance
indicated that the tip was lodged in the anterior cerebral artery and
thus blocked blood flow to the MCA. Reperfusion was induced in
group I by removing the suture 90 minutes after MCAO
Complications and Pitfalls in Rat Stroke Models
T2-Weighted Imaging
High-resolution multislice proton-weighted and T2-weighted
double-contrast spin-echo imaging was used to map lesion and
hemispheric volumes. Sixteen contiguous coronal slices (thickness,
2 mm) were acquired with a field of view of 37⫻37 mm and a matrix
size of 512⫻256 [repetition time (TR)⫽3000 ms, echo time 1
(TE1)⫽27 ms, echo time 2 (TE2)⫽72 ms, imaging time⫽25.5 min,
2 averages].
Computer-aided planimetric assessment of the lesion and hemispheric volumes were performed using image analysis software
(Image J 1.25s; National Institutes of Health). After adjustment of
contrast, the contours of the hemispheres were traced manually on
each slice. The position of the midline was determined using
neuroanatomic landmarks as described previously.7 Lesion volumes
were determined by computer-aided manual tracing of the hyperintense lesions and corrected for the space-occupying effect of brain
edema as described previously using the following equation7:
Macrosphere Model
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PE-50 tubing, filled with saline and 6 TiO2 macrospheres (diameter,
0.315 to 0.355 mm; BRACE GmbH, Alzenau, Germany), was
inserted into the ECA stump. The tip of the tubing was placed in the
carotid bifurcation without affecting the blood flow to the ICA. Then
the macrospheres were advanced separately into the ICA by a slow
injection of ⬇0.05 mL saline each, until they were moved passively
into the cerebral circulation by the blood flow. After macrosphere
injection, the ICA was carefully flushed with 0.5 mL saline.4
Experimental Protocol
Thirty animals were randomly subjected to group I to III: (1) group
I, MCAO for 90 minutes, followed by reperfusion (suture model;
n⫽10); (2) group II, permanent MCAO (suture model; n⫽10); and
(3) group III, permanent MCAO (macrosphere model; n⫽10).
The following exclusion criteria were used: no neurological
deficits 3 hour after MCAO (score 0); occlusion of the MCA main
stem on MRA after 24 hours in the reperfusion group; MCA main
stem not being occluded on MRA after 24 hours in groups II and III;
no infarction in the MCA territory on T2-weighted MRI and
2,3,4-triphenyltetrazolium chloride (TTC) staining; subarachnoid
hemorrhage (SAH) on postmortem examination; hypothalamic infarction with subsequent hyperthermia (in group III only). All
excluded animals were replaced.
Three additional animals per group were subjected to MCAO to
determine physiological parameters. Blood pressure, blood gases,
and glucose were measured 10 and 100 minutes after MCAO and
were compared with baseline values (5 minutes before MCAO).
Clinical Evaluation
2373
%HLVe⫽
HCc⫺HVi⫹LVu
䡠 100
HVc
where %HLVe indicates edema– corrected lesion volume (in percent
of the hemispheric volume); HVc and HVI indicate contralateral and
ipsilateral hemispheric volume; and LVu indicates uncorrected lesion
volume.
MRA
Arterial MRA was acquired using a 2-dimensional time-of-flight
sequence. Sixty contiguous coronal slices (thickness, 0.3 mm) were
acquired with a field of view of 37⫻37 mm and a matrix size of
256⫻256 (TR⫽25 ms, TE⫽5 ms, TA⫽25.5 min, flip angle⫽90°, 3
averages), and a moving saturation slice of 20-mm thickness at the
cranial side of the measured slice for suppression of venous signal.
Three-dimensional reconstruction (maximum intensity projection)
was performed with use of the Image Processing Tool of the
Paravision 2.1 software (Pharmascan) after interpolation to isotropic
voxel size.
Postmortem Analysis
After MRI, the animals were deeply anesthetized and euthanized.
The brains were removed and the localization of the macrospheres in
the basal cerebral arteries was determined using a magnifying glass.
The brains were sectioned coronally into 6 slices (thickness, 2 mm),
incubated in a 2% solution of TTC at 37°C, fixed by immersion in
10% buffered formalin, and scanned with a computer scanner
(resolution 600⫻600 dpi). Using image analysis software (ImageJ),
the areas of the ipsilateral hemispheres and of the infarcted regions
were calculated. Lesion volume was corrected for the spaceoccupying effect of brain edema using the following equation:
Neurological evaluation was performed 3 hours and 24 hours after
induction of ischemia and scored on a 7-point scale: 0, no neurological deficit; 1, failure to extend left forepaw fully; 2, inconstant
circling to the left; 3, constant circling to the left; 4, falling to the left;
5, no spontaneous walking with depressed level of consciousness;
and 6, death.
MRI
After clinical evaluation at 24 hours, the animals were placed in an
MRI-scanner (Bruker PharmaScan 7.0T, 16 cm). Respiratory rate
was monitored during the imaging protocol. Isoflurane concentration
was varied between 2.0% and 3.0% to keep the respiratory rate
between 60 and 80/min. Temperature was maintained at 37°C.
The linear polarized volume resonator (diameter, 60 mm) was
tuned and matched manually.
The MRI tomography machine operates at 300.51 MHz for 1H
imaging and is equipped with a 300-mT/m self-shielding gradient
system. Localizer images were acquired using a spin-echo sequence.
RARE sequences (20 contiguous slices, 1-mm thickness, TR⫽2500
ms, TE⫽41.8 ms) were used to verify symmetric positioning and
were repeated after correction of slice angulation, if necessary.
Figure 1. Time course of body temperature. Body temperature
increased in the permanent suture MCAO group only (group II).
In groups I and III, temperature remained within the normal
range.
2374
Stroke
TABLE 1.
October 2004
Clinical Findings, Lesion Volume, and Model Failure Rate
Score at
3h
Score at
24 h
Temperature
at 3 h
Temperature
at 24 h
%HLV at
24 h
Model Failure
Rate
37.4°C⫾0.7
37.2°C⫾0.5
14.6%⫾12.4
1/11 rats (9%)
38.8°C⫾0.5
38.0°C⫾0.5
35.2%⫾11.4
4/14 rats (29%)
37.4°C⫾0.4
37.3°C⫾0.5
21.3%⫾17.2
2/12 rats (17%)
Group I: suture (reperfusion), mean⫾SD
1.9⫾1.2
2.1⫾1.0
Median (range)
1 (1–4)
2.5 (1–3)
Group II: suture (permanent), mean⫾SD
2.1⫾0.9
1.9⫾0.9
Median (range)
2 (1–3)
2 (1–3)
Group III: macrosphere (permanent), mean⫾SD
2.0⫾1.1
2.0⫾1.1
Median (range)
2 (1–3)
2 (1–3)
Clinical scores did not differ among the groups or between the time points (P⬎0.05).
Compared to groups I and III, body temperature was significantly higher in group II 3 hours (P⬍0.01) and 24 hours (P⬍0.01) after MCAO.
Lesion volume was significantly smaller in the reperfusion group, compared to groups II and III.
The difference between groups II and III was not significant (P⬎0.05).
Model failure rate did not differ significantly among the 3 models (P⬎0.05).
%HLV indicates percent hemispheric lesion volume.
%HLV⫽LV/HVi
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where %HLV indicates edema– corrected lesion volume (in percent
of the hemispheric volume); LV indicates direct lesion volume; and
HVI indicates ipsilateral hemispheric volume.
Hypothalamic damage was determined from the TTC-stained
slices as described previously.8 Determination of infarct size and
hypothalamic injury was performed by an experienced investigator,
blinded to group assignment and clinical assessment.
Statistics
Data are presented as mean⫾SD. Parametric and nonparametric data
were compared using Student t test or Mann–Whitney U test. Model
failure rate was compared among the groups using Pearson ␹2 test.
P⬍0.05 was considered statistically significant.
Results
Clinical Findings
Body weight and clinical scores at 3 hours and at 24 hours
after MCAO did not differ among the groups (P⬎0.05). Body
temperature remained stable in groups I and III but increased
significantly in group II (P⬍0.01, compared with group I and
III) (Figure 1, Table 1).
MRI Findings
Occlusion of the MCA main stem could clearly be demonstrated
on MRI (Figure 2). Mean ischemic lesion size was significantly
smaller in the reperfusion group compared with groups II
(P⬍0.01) and III (P⬍0.05). Animals subjected to macrosphere
MCAO had smaller lesion volumes compared with animals
subjected to permanent suture MCAO (group II). This difference, however, was not statistically significant (P⬎0.05).
Physiological Parameters
Blood pressure, pO2, pCO2, pH, and glucose measured 10 and
100 minutes after MCAO were within the physiological range
and were not significantly different from baseline values or
among the groups (P⬍0.05; data not shown).
TTC Findings
Edema– corrected lesion volumes correlated well between
MRI and TTC staining (group I, r⫽0.95; group II, r⫽0.69;
group III, r⫽0.87).
Figure 2. MCAO techniques. Top, Suture
technique. After permanent MCAO,
MRA (left) indicates occlusion of the ICA
and MCA. The suture furthermore
occludes the HA and the AChA that originate from the ICA (middle). Brain infarction in the territory of the MCA, AChA
and HTA is clearly visible on
T2-weighted imaging after 24
hours (right). Bottom, Macrosphere technique. After permanent MCAO, MRA (left)
indicates occlusion of the MCA, but not
of the proximal part of the intracranial
portion of the ICA. The spheres block
blood flow to the MCA and to the AChA,
but not to the HA. This leads to brain
infarction in the MCA and AChA territory,
but not in the hypothalamic region (MRI,
right). ACA indicates anterior cerebral
artery; BA, basilar artery; AChA, anterior
chorid artery.
Gerriets et al
TABLE 2.
Complications and Pitfalls in Rat Stroke Models
2375
Excluded Animals
Score at
3h
MRA
T2-weighted MRI
Postmortem Inspection
Temperature
TTC Staining
Group I
(suture, reperfusion)
1
MCA patent
MCA territory lesion
Subarachnoid hemorrhage*
Normal
MCA territory lesion
Group II (suture,
permanent occlusion)
2
MCA patent*
Small lesion, suggestive of
AChA and hypothalamic
artery infarction
Normal
Hyperthermia
Small lesion, suggestive of
AChA and hypothalamic
artery infarction
1
MCA patent*
Small lesion, suggestive of
AChA and hypothalamic
artery infarction
Normal
Hyperthermia
Small lesion, suggestive of
AChA and hypothalamic
artery infarction
3
MCA occluded
MCA territory lesion
Subarachnoid hemorrhage*
Normal
MCA territory lesion
0*
MCA patent
No ischemic lesion
Normal
Normal
No ischemic lesion
3
MCA occluded
Lesion in the territory of
the MCA and the
hypothalamic artery*
Spheres occluded proximal
ICA and MCA orifice
Hyperthermia
Lesion in the territory of
the MCA and the
hypothalamic artery*
0*
MCA patent
No ischemic lesion
Doubled MCA main stem;
spheres occluded only 1
main stem
Normal
No ischemic lesion
Group III (macrosphere,
permanent occlusion)
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Seven animals (18.9%) were excluded.
Clinical findings, MRI, and MRA data are presented for the individual animals.
MCA indicates middle cerebral artery; ICA, internal carotid artery.
*Reason for exclusion.
Model Failure Rate
Seven animals had to be excluded because of the prespecified
exclusion criteria and were replaced (Table 2):
Two animals (1 each in group II and III) did not show
neurological deficits 3 hours after MCAO. TTC staining
revealed no ischemic lesion after 24 hours. Inspection of the
basal cerebral arteries revealed an anatomic variant in 1
animal (group III) with a doubled MCA main stem.
A patent MCA main stem was detected on MRA in 2
animals subjected to permanent MCA suture occlusion. In
these animals, small subcortical lesions were detected on
MRI, suggestive of an infarction in the territory of the
anterior choroid artery (AChA) (Figure 3). Both animals
showed neurological deficits.
One animal in group III displayed additional ischemic
lesion in the territory of the HA, resulting in hyperthermia.
SAH was detected in 2 animals on postmortem examination
(1 each in group I and II) (Figure 4).
Discussion
In the present study, 3 different endovascular MCAO techniques were compared. In each model, complications and side
effects occurred that need to be considered to avoid misinterpretation of the results.
Verification of MCAO
Endovascular models can fail to occlude the MCA main
stem.1,10 These animals need to be identified and excluded
from studies. In the present study, we used a combination of
early clinical evaluation and time-of-flight MRA to confirm
MCAO.
MRA
Time-of-flight MRA allows the assessment of the intracranial
vascular status in rodents noninvasively without using contrast agents. MRA thus is noninvasive and can easily be
repeated (ie, to document sufficient MCAO and
reperfusion).9,10
In the present study, MRA provided easily visualized
imaging of the circle of Willis. Occlusion of the ipsilateral
MCA was easy to verify. As expected, the ipsilateral MCA
was patent in all rats in group I, indicating sufficient reperfusion after withdrawal of the suture. MCAO was confirmed
in all animals in group III and in all but 2 rats in group II
Figure 3. Model failure (permanent
suture MCAO). Inappropriate insertion
depths of the suture caused an occlusion of the AChA and the HA, but not of
the MCA (left/middle). T2-weighted MRI
revealed a small subcortical lesion, suggestive of AChA and HTA territory infarction (right). The animal showed moderate
to severe neurological deficits and
increased body temperature after 3
hours and 24 hours.
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Stroke
October 2004
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Figure 4. Model failure (subarachnoid hemorrhage). T2-weighted
MRI 24h after permanent suture MCAO. Vessel perforation,
caused by inappropriate suture insertion depths, resulted in
SAH and intracerebral hemorrhage (arrow).
(Figure 2). In the latter 2 animals, the suture technique
obviously failed to occlude the MCA. Thus, MRA appears to
be a reliable, noninvasive method to verify MCAO in
endovascular stroke models.
Neurological Evaluation
Many authors suggest excluding animals that do not show
clinical signs of focal cerebral ischemia (ie, hemiparesis or
circling), indicating insufficient vessel occlusion. In the
present study, 2 animals showed no clinical deficits after
MCAO and were therefore excluded. Lack of MCAO was
confirmed by MRA and normal TTC staining after 24 hours
in both rats.
In contrast, the suture technique in particular might fail to
occlude the MCA, although the animals show neurological
deficits. Two rats in group II displayed neurological deficits
(failure to extend left forepaw and inconstant circling) 3
hours after insertion of the suture. On MRA, however, the
MCA was patent in both rats. Careful inspection of the
T2-weighted images and TTC staining indicated small lesions, suggestive of ischemic damage in the territory of the
AChA but not in the MCA territory (Figure 3). We conclude
that in these particular animals the occluder had not advanced
deeply enough into the ICA, so the tip of the suture occluded
the AChA (which originates from the intracranial portion of
the ICA) but not the MCA.8,11
In conclusion, absence of clinical neurological deficits
might indicate that the model failed to occlude the MCA.
Presence of neurological deficits, however, does not provide
evidence for MCAO, and additional methods, such as MRA,
are desirable to confirm sufficient vessel occlusion.
Other Methods
As an alternative to MRA, laser Doppler monitoring1,12 or
perfusion-weighted imaging4,13 can be used to determine
sufficient MCAO and reperfusion. In contrast to MRA, both
methods provide (semi-)quantitative information about cerebral perfusion. These methods, however, have some disadvantages. Placement of the laser Doppler probes requires
prolonged anesthesia and trepanation of the skull, leading to
various side effects, such as reduction of intracranial pressure
and local brain temperature, imaging artifacts, and others,1
and thus might confound experimental stroke studies.
Perfusion-weighted imaging can lead to false-positive results
caused by occlusion of the ipsilateral ICA, which can reduce
perfusion in the MCA territory. Furthermore, perfusion imaging might indicate a reduced local cerebral perfusion in
case of AChA occlusion, despite the MCA being patent, and
thus lead to false-positive results (Figure 3).
Postmortem inspection of the circle of Willis theoretically
allows one to determine the correct position of the occluder
within the basal cerebral arteries. This procedure, however,
might not be reliable for the suture model (and thus has not
been performed in the present study), because dislocation of
the suture can occur during the process of decapitation and
removal of the brain. Using the macrosphere model, in
contrast, the site of intracranial vessel occlusion can be
determined reliably by visual inspection of the circle of
Willis, because the spheres are lodged tightly within the
arteries and cannot be dislocated by manipulation during the
process of decapitation and brain removal.4 – 6 In the present
study, we found a perfect agreement between MRA findings
and verification of MCA main stem occlusion based on visual
inspection of the circle of Willis in the macrosphere group.
Subarachniod Hemorrhage
SAH results from perforation of the intracranial portion of the
ICA or of the anterior cerebral artery and represents a typical
complication of the suture MCAO model.1,4,5,12 This complication was confirmed by MRI (Figure 4) and postmortem
examination in 1 out of 11 animals in group I (9%) and in 1
out of 14 rats in group II (7%). SAH can lead to serious side
effects such as vasospasm and intracerebral hemorrhage, and
thus can confound the results of experimental stroke studies.
Previous reports indicated that laser Doppler-guided placement of the suture might reduce the incidence of SAH.1,12
In the present study and in previously published experiments, no case of SAH was documented in animals subjected
to macrosphere MCAO.4,5,6 Thus SAH may not occur in this
model.
Hypothalamic Infarction and Hyperthermia
Previous studies indicated that hyperthermia is accompanied
by an ischemic lesion of the hypothalamic region. The
pathological increase in temperature after permanent suture
MCAO (group II) can be explained by obstruction of the HA
that originates from the distal portion of the ICA, because the
suture occludes the entire carotid artery from the bifurcation
to the origin of the MCA (Figure 2).8,11,14 Hyperthermia did
not occur if the suture was removed within the first 90
minutes after MCAO (group I).1,8,14
Hypothalamic damage and subsequent hyperthermia are
important complications in experimental stroke research. This
condition has been shown to nullify the neuroprotective effect
of the NMDA receptor antagonist MK-801 in permanent
MCAO.3,5 In the present study, hypothalamic damage and
pathological hyperthermia were constant findings in permanent MCAO using the suture technique (group II). Reperfu-
Gerriets et al
sion at 90 minutes (group I) and use of the macrosphere
technique for permanent occlusion (group III) efficiently
avoided this side effect (Figures 1 and 2). Hypothalamic
damage was detected in only 1 animal in group III which has
been excluded.
Complications and Pitfalls in Rat Stroke Models
3.
4.
Conclusion
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Failures and side effects occur frequently in endovascular
stroke models. In the present study, insufficient occlusion of
the MCA was documented in the suture model and in the
macrosphere technique. These animals cannot be identified
reliably on the basis of clinical examination, because some
rats display hemiparesis caused by ischemic lesions in the
territory of the AChA, although the MCA was not occluded.
Therefore, additional methods to verify MCAO are
warranted.
The present study proved MRA to be a reliable and
noninvasive method for the verification of MCAO. Based on
clinical findings alone, only 2 out of 4 animals with insufficient MCAO were identified. The 2 remaining animals would
have not been detected without the use of MRA.
SAH was detected in 2 out of 25 animals subjected to
suture MCAO. This side effect did not occur in the macrosphere model and appears to be a specific complication of the
suture techniques.
Hypothalamic infarction and pathological hyperthermia
could be verified in all animals subjected to permanent suture
MCAO. This side effect can be avoided by withdrawal of the
suture after 90 minutes or by using the macrosphere technique for permanent MCAO.
5.
6.
7.
8.
9.
10.
11.
12.
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