From the Second Department of Internal Medicine, Faculty of Medicine,
Kyushu University (H.C., H.Y., S.I., M.F.), Fukuoka; and the Center for
Emotional and Behavioral Disorders, Hizen National Hospital (H.Y., H.U.),
Saga, Japan.
Correspondence to Hong Cai, MD, Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-8582, Japan.
Methods—Twenty-four male and 8 female SHR/Kyushu, 15 male and 5
female SHR/Izm, and 6 male Wistar-Kyoto rats (WKY)/Izm (5 to 7 months
old) were subjected to photothrombotic distal MCA occlusion, and
infarct volumes were determined.
Results—Although blood pressure levels were essentially the same
between the two substrains of hypertensive rats, infarct volumes were
significantly larger in the SHR/Kyushu substrain than in SHR/Izm of
either sex (P<0.001); infarct volumes in male and female
SHR/Kyushu were 83.8±11.7 and 58.5±9.2 mm3, and
those in male and female SHR/Izm were 61.5±10.7 and 34.8±7.9
mm3, respectively (values are mean±SD). Male SHR/Kyushu
that had simple Y-shaped MCA showed smaller infarcts (75.8±14.6
mm3, n=11) than those with more branching (regular) MCA
(93.2±19.1, n=13), the difference being significant
(P=0.022). Male SHR/Izm with simple distal MCA also produced
smaller infarctions than those with regular MCA (51.0±3.7 versus
68.9±8.7 mm3, P=0.0004).
Conclusions—Photothrombotic occlusion of distal MCA in
hypertensive rats provides a simple and reproducible model of focal
ischemia. Most importantly, this study emphasizes the
substantial variabilities in infarct sizes caused by the differences in
substrains of SHR, gender, and distal MCA patterns.
The results of Duverger and MacKenzie5 revealed
considerable differences in lesion size and location, depending on such
factors as strain, arterial blood pressure, and blood
glucose concentration. Proximal MCA occlusion causes a small and
variable infarction in Wistar-Kyoto rat(s) (WKY), a large but
equally variable infarction in Sprague-Dawley rats, and the largest
and most consistent infarction in Fisher-344 rats, among
normotensive strains of each. In hypertensive rats, MCA occlusion gives
rise to much larger infarcts than in normotensive strains. Distal MCA
occlusion also produces the largest and most reproducible infarcts in
stroke-prone SHR (SHRSP).6 7 Recently, a thrombotic distal
MCA occlusion model of rats was established in earlier work and our
previous study.8 9 10 11 Photothrombotic infarcts produced by
distal MCA occlusion without CCA manipulation in SHR were
moderate-sized and localized reproducibly with an acceptable
coefficient of variation.11 12
Hypertensive rats are relevant to stroke research and are used widely
for studies of hypertension-related cerebrovascular complications.
However, one of the most confounding problems is that SHR or the
normotensive control WKY from different sources are genetically
heterogeneous.13 14 SHR/Izumo (Izm) have
emerged as a new "prototype" of SHR that is derived from original
SHR.13 14 15 SHR/Izm and its control WKY/Izm have a common
genetic marker pattern, a major histocompatibility antigen RT-1k type,
and are now commercially available. In the present study, we
attempted to assess the effects of substrain and gender differences in
hypertensive rats and of distal MCA anatomy on infarct size.
The present data are the first to show significant differences in
infarct volumes between two substrains of SHR.
Materials
Surgical Setup
Rats were mounted on a stereotaxic head holder in the
prone position, and a 2-cm incision was made vertically midway between
the right orbit and the right external auditory canal. The temporalis
muscle was separated and retracted, and, under an operating microscope,
a burr hole 3 mm in diameter was made 1 mm rostral to the
anterior junction of the zygoma and squamosal bone, revealing the
distal segment of MCA above the rhinal fissure. The dura was left
intact.
Distal MCA Patterns
Photothrombotic Distal MCA Occlusion
Infarct Size
Statistical Analysis
Infarct size in four different groups of rats were compared
(Figure 2
Pooled data of male hypertensive rats (24 SHR/Kyushu and 15 SHR/Izm)
were used to investigate the relationship between branching patterns of
distal MCA and infarct size. Infarct volume in SHR/Kyushu with simple,
Y-shaped MCA was 75.8±14.6 mm3, which was
<93.2±19.1 mm3 in other SHR/Kyushu with more
branching (regular) MCA (P=0.022) (Figure 3
The lower limit of CBF autoregulation is shifted to a higher
level and cerebrovascular resistance is increased in SHR compared with
normotensive rats.18 Furthermore, markedly decreased
cerebral perfusion pressure after bilateral carotid occlusions in SHR,
as represented by lowered carotid back pressure, resulted
in ischemic brain energy
metabolism19 20 and ischemic
infarction.21 These changes associated with long-standing
hypertension are considered to be major factors for the susceptibility
to global cerebral ischemia in SHR.22 In focal
ischemia, the narrower anastomoses between MCA and anterior or
posterior cerebral artery at cortical arterial boundary
zones in SHRSP compared with normotensive rats restrict blood flow into
the territory of the occluded MCA, resulting in large infarcts in
hypertensive rats.6 Our present results were
consistent with above studies, showing 2.7 to 3.7 times larger
infarctions in hypertensive rats (SHR/Izm and SHR/Kyushu) than in
normotensive WKY/Izm (22.7±3.6 mm3, n=6).
Sources of potential experimental variability in focal cerebral
ischemia models have been emphasized.23
Strain-related variables affect outcome in models of MCA
occlusion.24 25 We found intrastrain differences in
infarct volume in our distal MCA occlusion model. Because MABP levels
were the same between SHR/Kyushu and SHR/Izm, factors other than
hypertension probably account for different lesion sizes. Recent
linkage analyses have identified that blood
pressure–independent genetic factors, genetic foci on chromosome 1,
termed STR 1,26 or on chromosome 4,27
determine susceptibility to spontaneous stroke. Interestingly, the
former study showed that genetic foci on chromosomes 4 and 5 were
protective against stroke. Furthermore, a genetic focus (the QTL) on
chromosome 5 has been demonstrated to account for 67% of the variance
in infarct volume in SHRSP after occlusion of the MCA; there was no
linkage between this QTL and any known blood pressure
phenotypes.28 Such genetic factors, in addition to
long-standing hypertension, play critical roles in deteriorating
ischemic stroke.
The female SHRSP have a lower incidence of stroke and a longer life
span compared with males.29 In our experience, female SHR
have consistently lower blood pressure than male SHR and milder
disturbance in cerebral energy metabolism after
bilateral CCA occlusions.30 In the present study,
infarct size after MCA occlusion was smaller in female SHR than in
males. Lower blood pressure in female hypertensive rats may partly
explain the smaller infarcts in female than in male SHR. Although
female body weights are much less than those of males in SHR/Kyushu,
there was only a 5% difference in brain size between male and female
SHR/Kyushu. That is not enough to explain the 30% difference in
infarction volume. Ovarectomy deteriorates and estrogen attenuates
focal ischemic injury.31 32 Estrogen modulates the
vascular dysfunction by preserving nitric oxide synthesis in female
SHR.33 Therefore, two factors (ie, lower blood pressure
levels and gonadal hormones in female rats) may be responsible for the
gender difference in susceptibility to cerebral ischemia in
SHR.
During the course of establishing a criterion for laser irradiation of
the MCA in the distal field distribution, which is combined with an
infusion of photosensitizing dye rose bengal (ie, photothrombosis), we
found approximately one half of the distal MCAs in SHR are
simple.8 9 10 11 Infarct volume of SHR with simple Y-shaped
(Type I) MCA was smaller than that of the regular pattern (Types II,
III, IV, and V). Menzies et al34 failed to find any
relationship between the anatomical pattern of MCA and the surface size
of the brain infarction in Sprague-Dawley rats. The reason for the
discrepancy between the results by Menzies et al and our present
results is not clear, but our data suggest an important caveat to
pharmacoprotection studies; biased selection of SHR with simple or
regular distal MCA may generate misleading treatment effects.
In conclusion, this study demonstrates the critical importance of
several variables, such as differences in the substrain and gender
of hypertensive rats and arterial patterns of distal MCA,
in determining infarct volume in the simple model of photothrombotic
occlusion of distal MCA in SHR.
Received January 15, 1998;
revision received May 19, 1998;
accepted May 28, 1998.
Department
of Neurology,
University of Miami School of Medicine,
Miami, Florida
Why the simple MCA branching pattern should be less susceptible
to infarct development than the regular, multiple-branched pattern is
most intriguing. At first glance, this result would seem
counterintuitive, inasmuch as the more complicated regular pattern
should provide more avenues for retrograde blood infiltration via
collateral channels. However, as the authors have recognized,
anastomoses between neighboring territories in stroke-prone SHR are
narrower compared with normotensive rats (see reference 6, Cai et al).
Possibly this deficiency is compounded in SHR with the regular
branching pattern compared to SHR with the simple pattern, owing to the
presence of a larger proportion of higher-generation vessels (with
narrower diameters) in the former variety. Also, vasculature reflecting
the more complicated regular pattern may provide more vascular space
for blood-brain barrier breaches, which are known to result from
secretions produced during thrombosis.2 The fact that
these anastomoses are nonetheless effective to some degree is suggested
by noting that infarct volume resulting from the present method of
inducing a long MCA occlusion (reference 11, Cai et al) is about twice
that obtained by occluding the equivalent segment in three different
spots (reference 10, Cai et al).
Finally, it would be quite interesting to speculate which
branching pattern would be more susceptible to reperfusion injury, if
in fact reperfusion could be elicited in this model or in another one
utilizing a normotensive strain. In the case of SHR, this may be
difficult to observe inasmuch as ischemia alone confers
near-maximal damage to cerebral tissue3 ; evidently,
reperfusion injury can be seen only in the context of intermediate
ischemia, as moderated by the presence of sufficient collateral
channels. From this, one would predict that rats exhibiting the
single-branch simple MCA pattern would evince reperfusion injury at
early times, while those with more complicated MCA patterns would
likely already display irreversible damage at these same times.
Received January 15, 1998;
revision received May 19, 1998;
accepted May 28, 1998.
© 1998 American Heart Association, Inc.
Original Contributions
Photothrombotic Middle Cerebral Artery Occlusion in Spontaneously Hypertensive Rats: Influence of Substrain, Gender, and Distal Middle Cerebral Artery Patterns on Infarct Size
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Background and Purpose—To analyze the effects of
substrain and gender differences in spontaneously hypertensive rats
(SHR) and distal middle cerebral artery (MCA) branching patterns on
infarct size, we compared infarct volumes produced by photothrombotic
distal MCA occlusion using SHR/Kyushu and SHR/Izumo
(Izm).
Key Words: stroke, experimental • cerebral ischemia, focal • photochemistry • models, animal
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Reproducible animal models of stroke or focal
ischemic infarction are crucial to the study of the
pathophysiology of ischemic brain injury. Focal
ischemia models are relevant to the human clinical setting,
because ischemic stroke is the predominant type of
cerebrovascular disease. The subtemporal approach technique of Tamura
et al,1 2 occluding the proximal part of the middle
cerebral artery (MCA), has been established as a standard focal
cerebral ischemia model in the rat. Occlusion of the proximal
MCA in the rat is technically feasible, but this model is surgically
demanding. Chen et al3 used a method of more distal
occlusion of MCA above the rhinal fissure combined with permanent
ipsilateral and temporary contralateral common carotid artery (CCA)
occlusions. Brint et al4 also used a distal MCA occlusion
with permanent occlusion of the ipsilateral CCA and found great
variability in cortical infarct volumes in normotensive rat strains.
However, the infarcts were large and fairly consistent in
spontaneously hypertensive rat(s) (SHR).
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
All procedures were done in accordance with the Animal Care
Guidelines of Kyushu University.
A total of 58 rats (24 male and 8 female SHR/Kyushu, 15
male and 5 female SHR/Izm, and 6 male WKY/Izm) aged 5 to 7 months were
used in this study. SHR/Kyushu and SHR/Izm are two substrains of SHR.
SHR/Kyushu were maintained in the Kyushu University Animal Center under
specific pathogen-free conditions and fed regular rat chow (CLEA rodent
diet CE-2, containing 25% protein, Na+ 2.6 mg/g, and
K+ 10.6 mg/g per pellet) and tap water ad libitum. SHR/Izm
and WKY/Izm were obtained from a commercial vendor (Funabashi Farm,
Chiba, Japan) and commissioned by the Disease Model Cooperative
Research Association (Chiba, Japan) at the age of 3 months. Four groups
of rats (male and female SHR/Kyushu and SHR/Izm) were randomly assigned
to photothrombotic MCA occlusion (n=5 to 10 per group).
Rats were anesthetized with halothane (4% for
induction; 1.5% during the surgical preparation, with a face mask;
0.75% after intubation; and 0.5% for maintenance) in a
mixture of 70% nitrous oxide/30% oxygen. The right femoral artery and
vein were cannulated using PE50 tubing. The rats were endotracheally
intubated with PE240 tubing. Pancuronium bromide (an initial dose of
0.3 mg followed by 0.1 mg every 30 minutes) was
intravenously injected, and the rats were mechanically
ventilated. Mean arterial blood pressure (MABP) was
continuously monitored; physiological variables
were determined before and 1 and 2 hours after distal MCA occlusion.
Rectal and head temperature were maintained at 37.5°C and at 36.0°C
to 36.5°C, respectively, by means of a warming lamp.
The anatomy of the distal MCA was recorded by
drawing under an operating microscope (x20) through the cranial window
in 85 male SHR/Kyushu, 127 male Sprague-Dawley rats, and 42 male Wistar
rats and was classified into 8 categories (Figure 1
) based on observations by Rubino and
Young16 and Watson et al8 and our own
experience.11 We found that about half of the distal MCA
in male SHR were simple, and only a small number of male SHR had a
complicated distal MCA (Table 1). (In the
present study, the distal MCA pattern of female rats was not
analyzed because of insufficient numbers of rats.) Therefore,
the lower half of distal MCA of SHR can be enveloped with an
elliptical, almost linear, laser beam.
View larger version (12K):
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Figure 1. The 8 branching patterns of distal MCA suitable for
male SHR, Sprague-Dawley rats, and Wistar rats (adapted from Figure 2 
of reference 8). P, F, T, and Py indicate parietal, frontal, temporal,
and pyriform branches, respectively.
View this table:
[in a new window]
Table 1. Variations in Distal MCA of 3 Strains of Rats
A krypton laser operating at 568 nm (Innova 301, Coherent
Inc) was used to irradiate the distal MCA at a power of 20 mW. The
laser beam was focused with a 30-cm–focal length cylindrical lens (CKX
300, Newport Corporation) and positioned with a mirror onto the distal
MCA. The photosensitizing dye rose bengal (15 mg/mL in 0.9% saline;
Wako Pure Chemical Industries Ltd) was administered
intravenously to a body dose of 20 mg/kg over 90 seconds
simultaneous with 4 minutes of laser irradiation. Two hours
after distal MCA occlusion, the head wound was closed and the catheters
were removed. The rats were carefully weaned from the respirator and
returned to the home cage after regaining the ability to breathe
independently.
After 3 days, the rat was decapitated under amobarbital
anesthesia (100 mg/kg IP), and the brain was rapidly
removed. The entire brain was cooled in ice-cold saline for 10 minutes
and cut into 2-mm-thick coronal sections in a cutting block; the brain
slices were then immersed in 2%
2,3,5-triphenyltetrazolium chloride (Wako
Pure Chemical Industries Ltd) at 37°C for 30 minutes in the dark. The
posterior surface of each section was imaged via video camera, and the
infarct areas indicated by the lack of staining were calculated with
NIH Image software (version 1.56). The infarct volume of each rat was
calculated by the following formula (trapezoidal rule): V=d
[1/2(Y1+Yn)+Y2+...+Yi+...+Yn-1],
where V indicates volume; d, distance between the sections; and
Yi, cross-sectional area of i th section, and
where the ends (Y1 and Yn) are equal to
zero.17
The values were expressed as mean±SD. Differences in
physiological variables and infarct volume were
analyzed with the unpaired t test. The levels of
significance were set at P<0.0125 (Figure 2) and P<0.025 (Figure 3) according to the number of multiple
comparisons (ie, Bonferroni's principle).
View larger version (25K):
[in a new window]
Figure 2. Infarct volume in two substrains of SHR (male and
female SHR/Kyushu and SHR/Izm). Values are mean±S.D.
* P<0.001 vs SHR/Izm; # P<0.001 vs female
SHR/Kyushu and female SHR/Izm. According to Bonferroni's principle,
P<0.0125 was considered to be significant.
View larger version (30K):
[in a new window]
Figure 3. Infarct volume in male SHR/Kyushu (n=24) and
SHR/Izm (n=15) with simple or regular MCA patterns.
* P=0.022, ** P=0.0004 vs SHR/Kyushu (n=13) or
SHR/Izm (n=9) with regular MCA pattern. According to Bonferroni's
principle, P<0.025 was considered to be significant.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Physiological variables in experimental
groups are shown in Table 2.
Arterial gases were in the normal range. MABP averaged
183 mm Hg in male SHR/Kyushu, 180 in male SHR/Izm, 161 in female
SHR/Kyushu, and 155 in female SHR/Izm. Photothrombotic distal MCA
occlusion led to a consistent pattern of cortical infarction;
the coefficients of variation (SD divided by mean value) were 14% to
23%.
View this table:
[in a new window]
Table 2. Physiological Variables ). Although the levels of blood pressure were essentially the
same between male SHR/Kyushu and SHR/Izm and between female SHR/Kyushu
and SHR/Izm, mean infarct volume was larger in male SHR/Kyushu
(83.8±11.7 mm3) than in SHR/Izm (61.5±10.7
mm3) (P<0.001) and in female SHR/Kyushu
(58.5±9.2 mm3) than in SHR/Izm (34.8±7.9
mm3) (P<0.001). In female SHR/Kyushu and
SHR/Izm, the MABP levels were lower, and infarct volumes were smaller
than those in males (P<0.001). The difference of infarction
volumes was 30% between male and female SHR/Kyushu. Mean hemispheric
size determined on the fourth brain section in female SHR/Kyushu
(58.1±2.2 mm2) was 5% less than in males
(61.1±3.3 mm2) (P<0.05). Average-sized
cortical infarction in each group is presented in Figure 4.
View larger version (34K):
[in a new window]
Figure 4. Diagrams showing the representative
infarcts in male and female SHR/Kyushu and SHR/Izm. ). In male
SHR/Izm, simple distal MCA also produced smaller infarction than did
regular distal MCA (51.0±3.7 vs 68.9±8.7 mm3,
respectively, P=0.0004). In these analyses,
physiological variables (MABP, body weight,
arterial gases, hematocrit, blood glucose, and head
temperature) were not different between the groups of rats with simple
or regular MCA patterns.
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Although blood pressure levels were not different between the two
substrains of hypertensive rats (ie, SHR/Kyushu and SHR/Izm), infarct
volumes were significantly larger in the SHR/Kyushu substrain than in
SHR/Izm of either sex. In female SHR/Kyushu and SHR/Izm, MABP was lower
and infarcts were smaller than in male rats. Infarct volumes in male
SHR/Kyushu and SHR/Izm with simple Y-shaped MCA were smaller than those
with more branching (regular) MCA.
Acknowledgments
This study was supported in part by the Social Insurance
Agency Contract Fund commissioned by the Japanese Health Sciences
Foundation. We thank Toru Nabika, MD, and Brant D. Watson, PhD, for
their valuable advice during the course of this study.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Editorial Comment
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
By means of the photothrombotic method developed by Yao et
al (see reference 11), in which a 2-mm-long segment of the distal MCA
is occluded (in the absence of concurrent carotid artery occlusion),
Cai and coauthors in the accompanying article confirmed for the first
time that MCA territory infarct volume in SHR (Kyushu and Izumo
strains) is sensitive to gender1 and strain and,
strikingly, also sensitive to the MCA branching pattern but was not
influenced by differences in blood pressure between strains. The single
branch ("simple") Y-shaped MCA distribution yielded smaller
infarcts than the multiple branch ("regular") MCA pattern in each
strain-matched case, and this aspect (as the authors have suggested)
must now be taken into account to evaluate properly the results of drug
therapies on lesion development. Evaluation of drugs should also be
more feasible with the moderate infarct volumes produced by the
present method, because the resultant ischemia is
submaximal in severity (see reference 11, Cai et al). As long as
infarct volume remains the universal standard for such evaluations, the
findings of Cai and coworkers constitute an important contribution to
research in stroke development and its mitigation.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
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