Temperature-Regulated Model of Focal Ischemia in the Mouse: A Study With
Histopathological and Behavioral Outcomes
Philip A. Barber, Lisa Hoyte, Frederick Colbourne and Alastair M. Buchan
Stroke. 2004;35:1720-1725; originally published online May 20, 2004;
doi: 10.1161/01.STR.0000129653.22241.d7
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2004 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://stroke.ahajournals.org/content/35/7/1720
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office.
Once the online version of the published article for which permission is being requested is located, click
Request Permissions in the middle column of the Web page under Services. Further information about this
process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Stroke is online at:
http://stroke.ahajournals.org//subscriptions/
Downloaded from http://stroke.ahajournals.org/ by guest on March 5, 2014
Temperature-Regulated Model of Focal Ischemia
in the Mouse
A Study With Histopathological and Behavioral Outcomes
Philip A. Barber, MB ChB, MRCP(UK); Lisa Hoyte, BSc;
Frederick Colbourne, PhD; Alastair M. Buchan, BM FRCP
Background and Purpose—The importance of mouse stroke models has increased with the development of genetically
manipulated animals. We hypothesized that immediate postischemia hypothermia may attenuate ischemic brain injury
in the mouse.
Methods—Intraabdominal radio frequency probes were implanted in animals and core temperature monitored. Groups
included: MCAO-45-REG (45 minutes middle cerebral artery occlusion [MCAO]) temperature-controlled in the
postischemic period ⬎34°C for 24 hours; MCAO-45 (45 minutes MCAO) were allowed to self-regulate core
temperature during recovery; MCAO-30-REG (30 minutes MCAO), with the same temperature protocol as MCAO45-REG; MCAO-30 (30 minutes MCAO), with temperature protocol the same as MCAO-45. Behavior and histological
score was assessed at 7 days. The qualitative histological score assessed for injury in 18 specified regions.
Results—MCAO-45-REG core temperature (mean 34.94°C⫾0.8°C) was significantly different than the self-regulating
(MCAO-45, mean 33.1°C⫾2.3°C) for the first 4 hours after anesthesia (P⬍0.01). There was a trend toward similar
differences in temperatures for MCAO-30-REG and MCAO-30 (P⫽0.08). At 7 days, a greater improvement in behavior
score was observed for MCAO-45 and MCAO-30 compared with MCAO-45-REG and MCAO-30-REG (P⬍0.001).
The histological score confirmed reduced injury in unregulated temperature groups (MCAO-45-REG mean 38⫾10 and
MCAO-45 30⫾5.1, P⬍0.05; MCAO-30-REG 41⫾10 and MCAO-30 30⫾9, P⬍0.05).
Conclusions—Hypothermia is an important confounder of stroke injury in the intraluminal filament mouse model. Future
mouse stroke studies must use strict temperature regulation. (Stroke. 2004;35:1720-1725.)
Key Words: ischemic attack, transient 䡲 hypothermia 䡲 behavior
D
espite extensive animal research on the pathophysiology
of stroke, very little has been translated into clinically
effective therapies. Among the many clinical trials, only a
few have been “positive.” There is growing evidence that
revascularization therapies are efficacious clinically, reducing
neurological disability,1 illustrating that these approaches can
translate from the laboratory to the bedside. In contrast,
cytoprotectants effective in rodents have failed in human
clinical trials.2 The most commonly cited failings of preclinical studies include poor physiological monitoring, short
outcomes, and an absence of behavioral endpoints.3,4
To date, hypothermia is the only stroke treatment that has
translated to humans.5 Hypothermia used after cardiac arrest
improved functional and cognitive recovery. The effect of
mild hypothermia in reducing injury after global ischemia,
and infarct volume in transient focal ischemia is wellestablished in the laboratory.6,7
Mouse stroke models are being used increasingly to research complex molecular processes after ischemia. Exciting
developments concerning neuroprotective strategies have involved the genetic manipulation of endogenous enzymes that
may play a role in the progression of ischemic injury.
However, little is known about temperature regulation in
mouse strains after focal ischemia. This may be very important because hypothermia or hyperthermia affects ischemic
brain injury after middle cerebral artery occlusion (MCAO)
in animals.8 There are many studies using transgenic or
gene-depleted mouse species but none has controlled temperature in the postischemic period.9 –15 Another limitation of
these studies was a failure to assess long-term and functional
outcome because substantial cell death may progress over
time and histology does not always predict functional
outcome.16
Preliminary observations suggested that transient hypothermia occurred postischemia in the transient MCAO in the
mouse, and we hypothesized that this postischemia hypothermia profoundly mediates ischemic brain injury. In this study,
Received January 6, 2004; final revision received March 10, 2004; accepted March 30, 2004.
From the Department of Clinical Neurosciences (P.A.B., L.H., A.M.B.), University of Calgary, Calgary, Edmonton, Canada; and Department of
Psychology and Centre for Neuroscience (F.C.), University of Alberta, Edmonton, Canada.
Correspondence to Dr Philip A. Barber, Department of Clinical Neurosciences, University of Calgary, Institute for Biodiagnostics (West), Room 153,
3330 Hospital Drive, Calgary, AB T2N 4N1 Canada. E-mail [email protected]
© 2004 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000129653.22241.d7
1720
Downloaded from http://stroke.ahajournals.org/
by guest on March 5, 2014
Barber et al
we assessed the effect of controlling core temperature during
and after focal ischemia in the mouse on both behavior and
histological injury at 7 days. We describe the use of telemetry
to regulate core temperature in the postischemic mouse.
Materials and Methods
Animals
All experiments and procedures were approved by the local animal
care committee and were in line with the Canadian Council of
Animal Care guidelines. Male C57 Black 6 mice (3 months old, 25
to 35g; Charles River Breeding Laboratories, Ontario, Canada) were
prepared for transient MCAO using the intraluminal filament method.17 A total of 54 animals were used: 48 in 4 experimental groups
and 6 to determine blood pressure and arterial blood gases. The 4
groups included: MCAO-45-REG (45 minutes MCAO, N⫽14)
temperature-controlled throughout surgery for 2 hours at 36.5°C, and
subsequently regulated at ⬎34°C for 24 hours; MCAO-45 (45
minutes MCAO, N⫽14) temperature-controlled during surgery as in
MCAO-45-REG, after which animals self-regulated core temperature; MCAO-30-REG (30 minutes MCAO, N⫽10), with core temperature regulation as in MCAO-45-REG; MCAO-30 (30 minutes
MCAO, N⫽10), with temperature protocol the same as MCAO-45.
The threshold temperature of 34°C is consistent with the normal
minimum daytime diurnal temperature. Animal weight was recorded
before surgery and daily thereafter. Core temperature and blood
pressure were monitored as described.
Transient Focal Ischemia
Anesthesia was induced with isoflurane (3% initial, 1% to 1.5%
maintenance) in O2 and air (80%:20%). Briefly, under the operating
microscope, the left common carotid artery (CCA), the left external
carotid artery (ECA), and the left internal carotid artery (ICA) were
isolated and a 6-0 suture was tied at the origin of the ECA and at the
distal end of the ECA. The left CCA and ICA were temporarily
occluded. The silicon-coated nylon suture was introduced into the
ECA and pushed up the ICA until resistance was felt and the filament
was inserted ⬇9 to 10 mm from the carotid bifurcation, effectively
blocking the middle cerebral artery (MCA). The diameter of the tip
of coated suture was considered acceptable between 180 and 220
micrometers.18 The suture remained inserted for 30 or 45 minutes,
after which it was removed and the ECA was permanently tied.
Subcutaneous normal saline (0.9%) was administered daily, adjusting the volume according to the animal’s weight loss. We estimated
that the daily requirement for water was 2 mL, and if the weight
decreased by 1 gram over 24 hours an additional 1 mL of 0.9% saline
was administered. This was continued until the animal’s weight
increased to within 5% of baseline.
Measurement of Cerebral Blood Flow
Transcranial measurements of cerebral blood flow (CBF) were made
by laser-Doppler flowmetry (LDF). A 0.5-mm diameter microfiber
laser-Doppler probe (Probe 418; Perimed) was attached to the skull
with cyanoacrylate glue 6 mm lateral and 1 mm posterior of bregma.
While under general anesthesia, regional cerebral blood flow (rCBF)
was monitored within the infarct core and in the parietal cortex
(penumbra). The surgical procedure was considered adequate if
ⱖ70% reduction in rCBF occurred immediately after placement of
the intraluminal occluding suture; otherwise, mice were excluded.
Blood flow velocity was measured preischemia, during MCAO and
reperfusion, using data collection software (Perisoft Version 1.3;
Perimed Inc). Data were expressed as a mean percentage of the
baseline preischemia value.
Temperature Regulation
Mice were implanted with intraabdominal radiofrequency probes
(TA10TA-F20; Transoma Medical) 7 days before MCA occlusion.
Core temperature was sampled every 30 seconds using receivers
(RLA-1020; Data Sciences Int) interfaced to a computer running
Temperature-Controlled Mouse Study
1721
ART 2.2. This telemetry system minimizes stress and allows
temperature monitoring/control in the freely moving animal.6 Core
temperature and activity (counts per 30 seconds) was initially
monitored for 7 days (N⫽15). The movement of the animal over the
receiver is detected as activity (via changes in the probe’s signal
strength). Thus activity is a relative measure and does not indicate an
exact distance traveled. During the surgical procedure, the animals
were regulated (total time 2 hours) by a 125-W heating lamp.
Blood Pressure and Arterial Blood Gas Analysis
The ventral surface of the tail was incised to expose the artery (N⫽3
additional animals in each group). The tail artery was isolated and cannulated with modified PE 10 tubing connected to a pressure transducer. Blood
pressure was monitored continuously, but was recorded every 10 minutes
before surgery, during ischemia, and after reperfusion. One sample of
arterial blood (0.3 mL) was acquired immediately after reperfusion for
determination of pO2, pCO2, pH, and glucose.
Behavior
Mice were assessed 4 hours after anesthesia and at 7 days. Behavioral assessment consisted of scoring: forelimb flexion, reduced
resistance to lateral push, gait toward the paretic side, and rotational
behavior.1920 The scoring was performed by an individual unaware
of the treatment condition (L.H.). The score used was as follows: 0,
normal; 1, consistent forelimb and axial flexion toward the contralateral to lesion side when lifted by the tail; 2, observations of score 1
with consistently reduced resistance to lateral push and gait toward
the paretic side; 3, observations of score 2 plus large or weak circling
toward the paretic side; 4, score 2 plus small circling toward the
paretic side; and 5, score 2 plus tight circling toward the paretic side.
Histology
Animals were euthanized with sodium pentobarbital (70 mg/kg intraperitoneally) and transcardially perfused with 0.9% saline, followed by
4% paraformaldehyde. The brain was then embedded in paraffin;
sectioned at 6 ␮m thickness, and stained with hematoxylin and eosin. In
addition, consecutive sections were assessed for degree and extent of
cellular injury and pan-necrosis in a total of 18 regions through the
striatum (2 sections) and hippocampus (2 sections). The distance
between sections was ⬇1 mm. The specific regions include cortical
areas C1-C6; medial striatum (SM) and lateral striatum (SL); and
hippocampus (H). In each region, degree of neuronal injury was
assessed according to an incremental 5-point scale: 0 is normal; 1
denotes ⬍10% selective neuronal injury; 2 denotes 10% to 50%
neuronal injury; 3 denotes ⬎50% neuronal injury; and 4 indicates
confluent areas of pan-necrosis. The minimum total score was 0,
indicating a normal brain; a maximum score of 72 indicates pan-necrotic
tissue in all 18 areas (Figure 1). The grading system accounts for the
degree of injury at each site and the extent of the damage (ie, number of
sites affected). The histology was analyzed blinded to animal identity
and the interobserver reliability of the score was assessed between each
interpreter (P.A.B., L.H.). The histological assessment was also performed for the most anterior striatal slice (bregma 1.54 mm) and a more
posterior hippocampal slice (bregma ⫺3.52 mm), therefore including a
total of 6 slices. The cerebral infarct volume was calculated by
measuring the ipsilateral and contralateral hemispheric volume and
subtracting the difference (mm3), assuming that the contralateral hemisphere was unaffected by ischemia. This method was used because at 7
days, the infarct could not be clearly defined.
Statistics
For all experiments with parametric data, a paired t test was used for
comparison of continuous data. Mann–Whitney U test was used to
compare the means of ordinal data. One-way ANOVA was used for
the interobserver correlation coefficient between observers (P.A.B.,
L.H.). Comparisons of proportions were assessed using 2⫻2 tables
and exact methods. Spearman correlation coefficients were used to
determine the relationship of animal activity and temperature and of
histological injury and behavior. Data are presented as mean⫾SD,
with a value of P⬍0.05 considered to be significant statistically.
Downloaded from http://stroke.ahajournals.org/ by guest on March 5, 2014
1722
Stroke
July 2004
Blood Pressure and Arterial Blood Gas Measurements During
30 and 45 Minutes MCAO
45-Min MCAO
Mean⫾SD
BP (mm Hg)
101 (7.9)
30-Min MCAO
Mean⫾SD
106 (7.6)
P
0.42
BP postreperfusion (mm Hg)
102 (14.1)
99.7 (4.5)
0.75
pH (mm Hg)
7.32 (0.1)
7.31 (0.1)
0.70
PCO2 (mm Hg)
45.2 (10.2)
43.6 (8.3)
0.83
PO2 (mm Hg)
93.3 (17.7)
107.3 (2.5)
0.25
Hemoglobin (g/dL)
14.9 (0.2)
15.1 (0.4)
0.48
Glucose (mmol/L)
8.6 (1.7)
8.6 (1.5)
0.98
BP indicates blood pressure; Min, minute.
Figure 1. Qualitative histological grading system at 7 days. Histological sections (A and B) are examples of cerebral infarction
in MCAO-45-REG. Diffuse necrosis can be observed within the
striatum and cortical mantle.
Results
The mean weight of animals was 30.6⫾2.3 grams (MCAO45-REG: 30.4⫾2.6; MCAO-45: 30.6⫾2.6; MCAO-30-REG:
30.2⫾2.0; MCAO-30: 31.3⫾1.9; P⬎0.5). Cortical blood
flow measured using LDF decreased after MCA occlusion by
at least 70% in all animals. After insertion of the intraluminal
filament, the mean rCBF decreased to 11.5%⫾5.5% of the
preocclusion value. There was no difference statistically in
the intraischemic relative rCBF between MCAO-45-REG
(mean 10.1⫾4.9) and MCAO-45 (mean 13.9⫾5.6, P⫽0.07),
or between MCAO-30-REG (mean 11.4⫾6.1) and MCAO-30
Figure 2. Regional cerebral blood flow (rCBF) before ischemia,
during MCAO (30 and 45 minutes), and in reperfusion.
(mean 10.21⫾5.1, P⬎0.5). Immediately during reperfusion,
the LDF reading returned to 88.9%⫾26.7% of the baseline
preocclusion value. Similarly, there was no significant difference in the LDF recordings during reperfusion between
MCAO-45-REG (mean 81.2⫾27.33) and MCAO-45 (mean
100.5⫾32.8, P⬎0.1), or MCAO-30-REG (mean 91.6⫾19.7)
and MCAO-30 (mean 80.3⫾17.1, P⬎0.2). LDF measurements were recorded for 60 minutes after reperfusion (Figure
2) and confirmed a restoration of CBF to within 90% of
preocclusion values, which decreases dramatically, revealing
a relative hypoperfusion (mean values 30%). The blood
pressure recordings and blood gas analysis are in the Table.
Temperature was monitored for 7 days in free-moving
animals housed in a day/night light cycle (N⫽15). The mean
temperature during this period was 36.23°C⫾0.5°C (range
33°C to 38°C). The general activity (counts per 30 seconds,
averaged hourly and including 168 data points over 7 days)
correlated very highly with the temperature variation
(r⫽0.72, P⬍0.001) shown in Figure 3.
In Figure 4 the temperature recordings are shown for the
self-regulating animals (MCAO-45 and MCAO-30) and those
controlled at ⬎34°C (MCAO-45-REG and MCAO-30-REG).
MCAO-45-REG core temperature (mean 34.94°C⫾0.8°C)
was significantly higher than the unregulated temperature
Figure 3. Diurnal temperature and activity in mice (N⫽15) over 7
days.
Downloaded from http://stroke.ahajournals.org/ by guest on March 5, 2014
Barber et al
Temperature-Controlled Mouse Study
1723
Figure 4. Core temperature (°C) and
activity (counts per minute) after the surgical period (2 hours) in animals that
underwent transient MCAO (30 and 45
minutes).
groups (MCAO-45, mean 33.1°C⫾2.3°C) for the first 4 hours
after the end of surgical temperature regulation (P⬍0.05). A
different mean core temperature was observed between
MCAO-30-REG (mean 35.4°C⫾0.9°C) and MCAO-30
(mean 34.6°C⫾1.01°C), which was not significant
(P⫽0.088). Figure 4 also presents the relative animal activity
(counts per minute), and the reduced initial activity corresponds to reduced core temperature in groups MCAO-45 and
MCAO-30.
Behavioral deficits were recorded 4 hours after anesthesia
and at 7 days. In MCAO-45-REG and MCAO-45, the
behavior score was equivalent at 4 hours, but at 7 days an
improvement in behavior score was observed, which was
greater in the unregulated groups (Figure 5A). Similar results
are shown for MCAO-30-REG and MCAO-30. There were 4
animal deaths in MCAO-45-REG (compared with MCAO30-REG, P⬍0.05) and 3 for MCAO-45 (compared with
MCAO-30, P⫽0.06).
Histological sections were assessed for neuronal injury and
frank necrosis in the specified regions. The mean of the
scores (34.6⫾9.6, range 18 to 57) correlated with behavioral
outcome (r⫽0.46, P⫽0.003). The histological score and
infarct volume measurements confirmed reduced injury in
unregulated temperature groups (Figure 5B). The use of the
histological score of 6 slices (including bregma 1.54 and
⫺3.52) produced a similar result (P⬍0.01). All animals had
hypothalamic damage. The intraclass correlation coefficient
between histological raters was 0.88 (95% CI, 0.81 to 0.95)
and indicates excellent agreement.
Discussion
Mild hypothermia occurs after MCAO in the mouse, and this
hypothermia improves behavioral and histological outcome. The
fact that hypothermia confounds experimental results is not new
because hypothermia has confounded previous pharmacotherapeutic studies.8 For example, N-methyl-D-aspartate antagonists
(eg, MK-801) provide little or no protection when temperature is
maintained.21 Furthermore, the cytoprotection afforded by
NBQX, an AMPA antagonist, is caused by protracted
hypothermia.22
Insight into why mice become mildly hypothermic after focal
ischemia can be obtained by studying their temperature patterns
and the relationship to animal activity. The baseline diurnal
temperature strongly correlates to the animal’s activity. As their
daytime motor activity decreases, there is a decrease in the core
temperature. The converse is true at night when motor activity
and temperature increases. The daytime/nighttime core temperature differential is up to 4°C, a phenomenon not seen in gerbils
or rats (⬇1°C). The postischemic mouse is inactive for several
hours while thermoregulation is poorly maintained and heat is
lost through their relatively large surface area. In addition, we
found hypothalamic injury. This is probably caused by transient
occlusion of hypothalamic arterial perforators originating off the
internal carotid artery by the intraluminal filament.
Downloaded from http://stroke.ahajournals.org/ by guest on March 5, 2014
1724
Stroke
July 2004
was considered to partly explain variability in infarct volume.26
It is also worth mentioning that postischemic hypothermia has
also been documented in global cerebral ischemia.27 One additional alternative explanation is that the rCBF decreases because
of reduced metabolic demand by the injured brain tissue.
This study has some limitations. Long-term histological and
functional outcomes were not assessed. Most cell death evolves
rapidly within days if the insult is moderate to severe,17 but
substantial cell death may occur beyond this time frame if the
ischemia is mild28 or when a cytoprotectant is used.26,29 In
addition, we used a neurological deficit score, which was
initially developed in rats and modified for mice. These types of
neurological deficit score are not standardized and vary among
laboratories.
In summary, hypothermia confounds mouse studies of stroke
and can easily be controlled for with the use of telemetry
temperature probes. The study demonstrates that 7-day survival
can be achieved with consistent and reproducible histological
injury.
References
Figure 5. A, Behavioral outcome (NDS score) after transient
MCAO (30 and 45 minutes) (*P⬍0.001, **P⬍0.05). B, Histological injury score and infarct volume (mm3) for the same group of
animals as in A (*P⬍0.01, **P⬍0.05, ***P⬍0.01, ****P⬍0.05).
Connelly et al20 established the role of perioperative hypothermia on infarct volume and neurological outcome after
MCAO in mice. By using a biofeedback incubator system,
animals were kept normothermic for 90 minutes postischemia
and exhibited larger infarct volumes and more severe neurological outcomes. There are 2 potential drawbacks to their temperature monitoring. Firstly, such a system does not control hypothermia in the awake animal, which improves outcome in rats.6,7
Secondly, it is likely that prolonged rectal temperature monitoring in the awake animal induces a significant stress response that
may aggravate cerebral injury.23
The occurrence of hypothermia with the intraluminal filament
model in mice may differ from focal ischemia in rats in which
hyperthermia24 has been reported under hypothalamic involvement in the rat. These observations in rats have differed in the
distal MCAO model via clip occlusion7 and the intraluminal
filament model.25 Some of the difference may be explained by
the type of stroke model (eg, hypothalamic involvement with the
intraluminal filament model) or may be related to the technique
used to monitor temperature as mentioned previously.24 The
relevance of brief postischemic hyperthermia exacerbating ischemic injury may be less important than the issue that rectal
temperature control may induce fever, supporting the use of
telemetry systems for temperature measurement and control.
Another source of variability is the vascular anatomy in
different mouse strains.21 In this regard, rCBF velocity measurements using laser Doppler can help account for vascular variability among mouse strains. In this study, we demonstrate
consistent return of rCBF to within 90% of the baseline preocclusion value, with a decrease of rCBF after 10 to 20 minutes
after reperfusion, with a tailing-off of ⬇30% of baseline rCBF.
This might be caused by either thrombosis in the MCA or
vasoconstriction in damaged vessel after transient MCAO, and it
1. National Institute of Neurological Disorders and Stroke rt-PA Stroke
Study Group. Tissue plasminogen activator for acute ischemic stroke.
N Engl J Med. 1995;333:1581–1587.
2. Gladstone DJ, Black SE, Hakim AM. Toward wisdom from failure:
lessons from neuroprotective stroke trials and new therapeutic directions.
Stroke. 2002;33:2123–2136.
3. Stroke Therapy Academic Industry Roundtable (STAIR). Recommendations for standards regarding preclinical neuroprotective and restorative
drug development. Stroke. 1999;30:2752–2758.
4. DeBow S, Clark DL, Maclellan CL, Colbourne F. Incomplete assessment
of experimental cytoprotectants in rodent ischemia. Can J Neurol Sci.
2003;30:368 –374.
5. Bernard SA, Gray TW, Buist MD, Jones BM, Silvester W, Gutteridge G,
Smith K. Treatment of comatose survivors of out-of-hospital cardiac
arrest with induced hypothermia. N Engl J Med. 2002;346:557–563.
6. Colbourne F, Li H, Buchan AM. Indefatigable CA1 sector neuroprotection with mild hypothermia induced 6 hours after severe forebrain
ischemia in rats. J Cereb Blood Flow Metab. 1999:742–749.
7. Colbourne F, Corbett D, Zhao Z, Yang J, Buchan AM. Prolonged but
delayed postischemic hypothermia: a long-term outcome study in the rat
middle cerebral artery occlusion model. J Cereb Blood Flow Metab.
2000;20:1702–1708.
8. Ginsberg MD, Busto R. Combating hyperthermia in acute stroke: a
significant clinical concern. Stroke. 1998;29:529 –534.
9. Kamii H, Mikawa S, Murakami K, Kinouchi H, Yoshimoto T, Reola L,
Carlson E, Epstein CJ, Chan PH. Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient
focal cerebral ischemia. J Cereb Blood Flow Metab. 1996;16:1153–1157.
10. Hara H, Fink K, Endres M, Friedlander RM, Gagliardini V, Yuan J,
Moskowitz MA. Attenuation of transient focal cerebral ischemic injury in
transgenic mice expressing a mutant ICE inhibitory protein. J Cereb
Blood Flow Metab. 1997;17:370 –375.
11. Sheng H, Bart RD, Oury TD, Pearlstein RD, Crapo JD, Warner DS. Mice
overexpressing extracellular superoxide dismutase have increased
resistance to focal cerebral ischemia. Neuroscience. 1999;88:185–191.
12. Rajdev S, Hara K, Kokubo Y, Mestril R, Dillmann W, Weinstein PR,
Sharp FR. Mice overexpressing rat heat shock protein 70 are protected
against cerebral infarction. Ann Neurol. 2000;47:782–791.
13. Sampei K, Mandir AS, Asano Y, Wong PC, Traystman RJ, Dawson VL,
Dawson TM, Hurn PD. Stroke outcome in double-mutant antioxidant
transgenic mice. Stroke. 2000;31:2685–2691.
14. Crack PJ, Taylor JM, de Haan JB, Kola I, Hertzog P, Iannello RC.
Glutathione peroxidase-1 contributes to the neuroprotection seen in the
superoxide dismutase-1 transgenic mouse in response to ischemia/
reperfusion injury. J Cereb Blood Flow Metab. 2003;23:19 –22.
15. Chen Y, Hallenbeck JM, Ruetzler C, Bol D, Thomas K, Berman NE,
Vogel SN. Overexpression of monocyte chemoattractant protein 1 in the
Downloaded from http://stroke.ahajournals.org/ by guest on March 5, 2014
Barber et al
16.
17.
18.
19.
20.
21.
22.
brain exacerbates ischemic brain injury and is associated with recruitment
of inflammatory cells. J Cereb Blood Flow Metab. 2003;23:748 –755.
Corbett D, Nurse S. The problem of assessing effective neuroprotection in
experimental cerebral ischemia. Prog Neurobiol. 1998;54:531–548.
Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral
artery occlusion without craniectomy in rats. Stroke. 1989;20:84–91.
Hata R, Mies G, Wiessner C, Fritze K, Hesselbarth D, Brinker G,
Hossmann KA. A reproducible model of middle cerebral artery occlusion
in mice: hemodynamic, biochemical, and magnetic resonance imaging.
J Cereb Blood Flow Metab. 1998;18:367–375.
Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski
H. Rat middle cerebral artery occlusion: evaluation of the model and
development of a neurologic examination. Stroke. 1986;17:472– 476.
Connolly ES Jr, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ. Procedural and strain-related variables significantly affect outcome in a
murine model of focal cerebral ischemia. Neurosurgery. 1996;38:
523–531.
Buchan A, Pulsinelli WA. Hypothermia but not the N-methyl-D-aspartate
antagonist, MK-801, attenuates neuronal damage in gerbils subjected to
transient global ischemia. J Neurosci. 1990;10:311–316.
Nurse S, Corbett D. Neuroprotection after several days of mild, druginduced hypothermia. J Cereb Blood Flow Metab. 1996;16:474 – 480.
Temperature-Controlled Mouse Study
1725
23. Clark DL, DeBow SB, Iseke MD, Colbourne F. Stress-induced fever after
postischemic rectal temperature measurements in the gerbil. Can
J Physiol Pharmacol. 2003;81:880 – 883.
24. Reglodi D, Somogyvari-Vigh A, Maderdrut JL, Vigh S, Arimura A.
Postischemic spontaneous hyperthermia and its effects in middle cerebral
artery occlusion in the rat. Exp Neurol. 2000;163:399 – 407.
25. Li H, Colbourne F, Sun P, Zhao Z, Buchan AM, Iadecola C. Caspase
inhibitors reduce neuronal injury after focal but not global cerebral
ischemia in rats. Stroke. 2000;31:176 –182.
26. Mao Y, Yang GY, Zhou LF, Stern JD, Betz AL. Focal cerebral ischemia
in the mouse: description of a model and effects of permanent and
temporary occlusion. Brain Res Mol Brain Res. 1999;63:366 –370.
27. Hossmann KA. Reperfusion of the brain after global ischemia: hemodynamic disturbances. Shock. 1997;8:95–101.
28. Garcia JH, Liu KF, Ye ZR, Gutierrez JA. Incomplete infarct and delayed
neuronal death after transient middle cerebral artery occlusion in rats.
Stroke. 1997;28:2303–2309.
29. Wang F, Corbett D, Osuga H, Osuga S, Ikeda JE, Slack RS, Hogan MJ,
Hakim AM, Park DS. Inhibition of cyclin-dependent kinases improves
CA1 neuronal survival and behavioral performance after global ischemia
in the rat. J Cereb Blood Flow Metab. 2002;22:171–182.
Downloaded from http://stroke.ahajournals.org/ by guest on March 5, 2014